CA2375033A1 - Vaccines against conformation-dependent antigens and against antigens that are not or are not only proteins or peptides - Google Patents
Vaccines against conformation-dependent antigens and against antigens that are not or are not only proteins or peptides Download PDFInfo
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- CA2375033A1 CA2375033A1 CA002375033A CA2375033A CA2375033A1 CA 2375033 A1 CA2375033 A1 CA 2375033A1 CA 002375033 A CA002375033 A CA 002375033A CA 2375033 A CA2375033 A CA 2375033A CA 2375033 A1 CA2375033 A1 CA 2375033A1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
Abstract
The invention relates to a method that makes it possible to use the highly effective technology of vaccination with deoxyribonucleic acid (DNA) not only on sequence epitopes of proteins or peptides, but also on conformation epitopes. The method also permits the use of DNA vaccination for antigens that are not or are only partially proteins or peptides. The preferred inventive vaccine contains a desoxyribonucleic acid (DNA) as its principal component.
This desoxyribonucleic acid codes for a peptide sequence which represents the immunological imitation (mimicry) of a conformation-dependent antigen including protein conformation epitopes or of an antigen that is not or is only partially a protein or peptide. The mimicry peptide, which is also or can also be part of the inventive vaccine, is either an antiidiotypic antibody, an antibody fragment, a peptide derived therefrom or a specifically binding peptide obtained by selection from a peptide gene bank. The invention can be used in medical and veterinary medical immunology, including in the adjuvant therapy of tumor diseases.
This desoxyribonucleic acid codes for a peptide sequence which represents the immunological imitation (mimicry) of a conformation-dependent antigen including protein conformation epitopes or of an antigen that is not or is only partially a protein or peptide. The mimicry peptide, which is also or can also be part of the inventive vaccine, is either an antiidiotypic antibody, an antibody fragment, a peptide derived therefrom or a specifically binding peptide obtained by selection from a peptide gene bank. The invention can be used in medical and veterinary medical immunology, including in the adjuvant therapy of tumor diseases.
Description
Vaccines against conformation-dependent antigens and against antigens that are not, or are not exclusively, proteins or peptides Description The invention refers to vaccines against conformation-dependent antigens and against antigens that are not, or are not exclusively, proteins or peptides. In addition, the invention refers to processes for their production and use as well as human antiidiotypical antibody fragments against the MUCl conformation epitope and amino acid sequences of mimicry peptides against the MUC1 conformation epitope as well as antiidiotypical antibody fragments against the TF antigen and amino acid sequences of mimicry peptides against the TF
carbohydrate epitope.
Target structures of vaccines against pathogens of infectious diseases and non-infectious diseases, including tumours, may be proteins or peptides, carbohydrates or lipids as well as combinations thereof. In the case of proteins and peptides, the immunogenic determinant (epitope) may be determined either by the sequence of the amino acids of a section of the molecule (sequential epitope) or by a specific arrangement of binding forces that do not correspond to the linear arrangement of the amino acids (conformation epitope). Conformation epitopes occur more frequently than sequential epitopes; mixed forms also occur.
Conformation epitopes and antigens that are not, or not exclusively, proteins or peptides are difficult to convert into an effective and practicable vaccine. As a rule, conformation epitopes only develop in native protein and not in shorter peptides. Antigens that are not, or not exclusively, proteins or peptides (such as glycostructures or lipids) are only slightly immunogenic. Their synthesis is often complicated.
A particularly serious fact is that, in many cases, these antigens are not correctly presented to the immune system.
However, an effective antigen presentation is, amongst other things, a prerequisite for the development of cytotoxic T
lymphocytes, i.e. for an effective cellular resistance.
Finally, the very effective form of DNA vaccination is not applicable to these antigens.
In DNA vaccination (genome vaccination), instead of a protein or peptide antigen, the coding DNA sequence itself, or packed in a vector, is injected as an intramuscular or intradermal vaccine. In this way, an effective humoral response and cellular response can be achieved (Wolff, J.A. et al., Science 247:1465, 1990; Ulmer, J.B. et al., Vaccine 12:1541, 1994; Raz, E. et al., Cancer res. 52:1954, 1992). A particularly successful procedure is what is known as the prime boost protocol (Keystone Symposia: DNA Vaccines, April 12 - 17, 1999, Snowbird, Utah, USA, conference tape) in which the intradermal, intramuscular or intrarectal inj ection of a DNA (priming) is followed by a booster with the corresponding antigen. A corresponding recombinant virus-vector particle (e. g. fowlpox, constructs derived from adeno or alpha virus) can also be used successfully. Of course, the prime boost procedure results in a strong cellular immune response with the activation of specific cytotoxic T cells, which is particularly desirable in tumour vaccines. The immune response can be significantly strengthened through the additional administration of suitable cytokines, also in the form of a DNA, through immunostimulating CpG-DNA motives (non-methylated cytosine-guanine-dinucleotides) or through suitable adjuvants (e. g. aluminium phosphates).
The object of the invention is to circumvent the aforementioned disadvantages and develop a vaccine, and more particularly a DNA
carbohydrate epitope.
Target structures of vaccines against pathogens of infectious diseases and non-infectious diseases, including tumours, may be proteins or peptides, carbohydrates or lipids as well as combinations thereof. In the case of proteins and peptides, the immunogenic determinant (epitope) may be determined either by the sequence of the amino acids of a section of the molecule (sequential epitope) or by a specific arrangement of binding forces that do not correspond to the linear arrangement of the amino acids (conformation epitope). Conformation epitopes occur more frequently than sequential epitopes; mixed forms also occur.
Conformation epitopes and antigens that are not, or not exclusively, proteins or peptides are difficult to convert into an effective and practicable vaccine. As a rule, conformation epitopes only develop in native protein and not in shorter peptides. Antigens that are not, or not exclusively, proteins or peptides (such as glycostructures or lipids) are only slightly immunogenic. Their synthesis is often complicated.
A particularly serious fact is that, in many cases, these antigens are not correctly presented to the immune system.
However, an effective antigen presentation is, amongst other things, a prerequisite for the development of cytotoxic T
lymphocytes, i.e. for an effective cellular resistance.
Finally, the very effective form of DNA vaccination is not applicable to these antigens.
In DNA vaccination (genome vaccination), instead of a protein or peptide antigen, the coding DNA sequence itself, or packed in a vector, is injected as an intramuscular or intradermal vaccine. In this way, an effective humoral response and cellular response can be achieved (Wolff, J.A. et al., Science 247:1465, 1990; Ulmer, J.B. et al., Vaccine 12:1541, 1994; Raz, E. et al., Cancer res. 52:1954, 1992). A particularly successful procedure is what is known as the prime boost protocol (Keystone Symposia: DNA Vaccines, April 12 - 17, 1999, Snowbird, Utah, USA, conference tape) in which the intradermal, intramuscular or intrarectal inj ection of a DNA (priming) is followed by a booster with the corresponding antigen. A corresponding recombinant virus-vector particle (e. g. fowlpox, constructs derived from adeno or alpha virus) can also be used successfully. Of course, the prime boost procedure results in a strong cellular immune response with the activation of specific cytotoxic T cells, which is particularly desirable in tumour vaccines. The immune response can be significantly strengthened through the additional administration of suitable cytokines, also in the form of a DNA, through immunostimulating CpG-DNA motives (non-methylated cytosine-guanine-dinucleotides) or through suitable adjuvants (e. g. aluminium phosphates).
The object of the invention is to circumvent the aforementioned disadvantages and develop a vaccine, and more particularly a DNA
vaccine, that can also be used in cases that, to date, were not suitable for such a vaccination.
The invention is realized according to the claims. On the one hand, it refers to a process by which the scope of application of the vaccination is expanded, and more particularly the DNA
vaccination, to conformation-dependent antigens and mixed forms (these also fall under the term conformation epitopes within the meaning of the invention), as well as antigens whose relevant epitopes are not, or not exclusively, proteins or peptides, e.g.
carbohydrates, combined carbohydrate-peptide epitopes, lipids, glucolipids, thus circumventing the aforementioned disadvantages. According to the invention, this is achieved through the detour via a peptide that is the immunological image of the original epitope (the antigen determinant) but whose amino acid sequence is different (mimicry peptide). The mimicry peptide is preferably obtained by using the well known methods of phage display or ribosome display (Scott, J.K. and Smith, G.P., Science, 249:386, 1990; Winter, G. et al., Annu Rev Immunol, 12:433, 1994; Hanes, J. et al., Proc Natl Acad Sci USA, 95:14130, 1998), either as a shorter peptide from peptide gene banks or in the form of an antiidiotypical antibody fragment from the respective gene banks. A third, more complicated method is to obtain antiidiotypical antibodies by means of the hybridome method. The common objective of the above three methods is to "rewrite" the original conformation epitope or the epitope that is not, or not exclusively, a protein or peptide into a corresponding immunological sequential epitope which permits a better immunological presentation and is suitable for a DNA vaccination. According to the invention, the vaccines, and more particularly the DNA vaccines, can be used not only in the form of the above example (prime boost protocol), but also in comparable variants and in the form of DNA vaccines alone or in mimicry structures alone and in suitable formulations.
The invention is realized according to the claims. On the one hand, it refers to a process by which the scope of application of the vaccination is expanded, and more particularly the DNA
vaccination, to conformation-dependent antigens and mixed forms (these also fall under the term conformation epitopes within the meaning of the invention), as well as antigens whose relevant epitopes are not, or not exclusively, proteins or peptides, e.g.
carbohydrates, combined carbohydrate-peptide epitopes, lipids, glucolipids, thus circumventing the aforementioned disadvantages. According to the invention, this is achieved through the detour via a peptide that is the immunological image of the original epitope (the antigen determinant) but whose amino acid sequence is different (mimicry peptide). The mimicry peptide is preferably obtained by using the well known methods of phage display or ribosome display (Scott, J.K. and Smith, G.P., Science, 249:386, 1990; Winter, G. et al., Annu Rev Immunol, 12:433, 1994; Hanes, J. et al., Proc Natl Acad Sci USA, 95:14130, 1998), either as a shorter peptide from peptide gene banks or in the form of an antiidiotypical antibody fragment from the respective gene banks. A third, more complicated method is to obtain antiidiotypical antibodies by means of the hybridome method. The common objective of the above three methods is to "rewrite" the original conformation epitope or the epitope that is not, or not exclusively, a protein or peptide into a corresponding immunological sequential epitope which permits a better immunological presentation and is suitable for a DNA vaccination. According to the invention, the vaccines, and more particularly the DNA vaccines, can be used not only in the form of the above example (prime boost protocol), but also in comparable variants and in the form of DNA vaccines alone or in mimicry structures alone and in suitable formulations.
In addition, the invention refers to vaccines against conformation-dependent antigens according to Claim 1. In the process according to the invention, using the phage display or the ribosome display method the relevant conformation epitopes are "rewritten" into a corresponding immunological sequential epitope that mimics the conformation epitope. The primary reagents used are molecules that specifically bind the target antigen in its desired conformation, e.g. antibodies, antibody fragments or receptors. Thus, from the various gene libraries, antibody fragments (antiidiotypical antibody fragments, Ab 2) or linear or circular peptides are obtained that specifically bind the primary reagents and immunologically mimic the antigen.
Alternatively, antiidiotypical antibodies are obtained by means of the hybridome method and fragments are isolated from them, if required. These mimicry peptides are rewritten into a DNA
and used as a DNA vaccine. One process is what is known as the prime boost protocol, in which the intradermal, intramuscular or intrarectal injection of a DNA (priming) in the form of a plasmid DNA, linear DNA or a plasmid replicon vector is followed by a booster with the corresponding antigen, alone, in the form of a chemical coupling of proteins, in the form of bacteriophages as fusion proteins with phage coat proteins on their surface, in the form of a fusion protein on the surface of other viruses or attenuated biological carriers or in the form of dendritic cells loaded with a peptide. In this case, the DNA as well as the expressed mimicry peptide are required, which is easy using the phage display or ribosome display method. Alternatively, a corresponding recombinant virus-vector particle (e. g. fowlpox, constructs derived from adeno or alpha virus) can be used successfully. The immune response can be significantly strengthened through the additional administration of suitable cytokines, also in the form of a DNA, through immunostimulating CpG-DNA motives (non-methylated cytosine-guanine dinucleotides) or through suitable adjuvants (e. g.
aluminium phosphates).
Besides vaccines against conformation-dependent antigens, the invention also refers to vaccines against antigens that are not, or not exclusively, proteins or peptides according to Claim 3.
A target antigen type of the group of antigens that are not, or not exclusively, proteins or peptides are glycostructures;
additional immunogenic structures are combined carbohydrate-protein epitopes, lipids, glycolipids or synthetic structures.
A process is known from DE 196 27 352 A1 with which a monoclonal antiidiotypical antibody can be obtained using the hybridome method, which immunologically mimics pure carbohydrate structures. According to the invention, starting with this antiidiotypical antibody, a vaccine (preferably a DNA vaccine of this antibody or a suitable fragment thereof) is used for the vaccination. Thus, the present invention expands several points of this process from DE 196 27 352 A1. Antiidiotypical antibody fragments can be obtained directly from the antibody gene libraries using the phage display method or the ribosome display method. Also with this process, human antibody fragments can be obtained directly. In addition, combined carbohydrate-peptide epitopes can also be used. Plus there is a process with which short linear or circular peptides which immunologically mimic the antigen (also known as mimicry peptides) can be obtained from peptide gene libraries, also using the phage display method or the ribosome display method. To this end, not only specific idiotypical antibodies (Abl) are used as primary reagents for the selection of these mimicking structures, but also other substances that specifically recognize the glycostructure, such as lectins or receptors. The process also includes the use of the obtained structures preferably as DNA
vaccines, alone or in conjunction with the antibodies that immunologically mimic the antigen, antibody fragments or peptides in a suitable formulation (see above and claims), for example in a suitable formulation of the prime boost protocol.
Furthermore, according to the invention the mimicking protein structures can also be used alone for vaccination.
The invention also refers to vaccines (in the full scope of the description for conformation-dependent antigens) against the antigens glycopeptides, glycolipids, lipids, synthetic structures or other antigens that are not, or are only partially, proteins or peptides, the relevant epitopes having improved immunological structures, as well as to their production processes and their use.
The immunotherapy approach to diseases involving tumours is based on the assumption that it is possible to strengthen or activate the natural immune response. The rationale for vaccination lies in combating the residual disease (metastasis prophylaxis) according to a conventional therapy (e. g. surgical removal of the main body of tumour cells). As the name implies, mimicry peptides immunologically mimic the original antigen or epitope. They do this to very high degree, but not completely.
This can be seen as positive for applications within the framework of a vaccine (and more particularly in the case of a tumour vaccine) in that specifically inhibiting processes, e.g.
tolerance phenomena, are circumvented.
The prerequisite for the development of defined tumour vaccines is not only the presence of tumour-specific antigens, but also knowledge thereof. Great progress has been achieved in this area during the past three decades, not least through the development of monoclonal antibodies.
One widespread cancer antigen is the epithelial mucin, MUC1, whose immune-dominant epitope occurs multiple times on the extracellular part of the molecule. In its native state, this epitope forms a type I-(3 turn, but on synthetic peptides only under certain conditions, e.g. when the theonin of the dominant immune region is glycosylated with GalNAca1-0-Thr or Gal~il-3GalNAca1-0-Thr (Karsten, U. et al., Cancer res 58:2541-2549, 1998). As a rule, this epitope is perceived as a typical conformation epitope by the immune system, see Example 1.
According to the invention, using the phage display method, this conformation epitope is mimicked by immunologically identical (or almost identical) sequential epitopes which, in the form of a DNA, are part of a tumour vaccine in a DNA vaccination vector (Example 1) .
Therefore, the object of the invention is also human antiidiotypical antibody fragments against the MUC1 conformation epitope as well as all DNA sequences that encode these fragments, and protein sequences or DNA or protein partial sequences that can be derived from them and that have the corresponding characteristics.
Primarily, this concerns the following human antiidiotypical antibody fragments against the MUC1 conformation epitope with the following sequence nos. 1 to 31.
Fragments that contain the desired DNA of the scFv and of the peptides were multiplied using the PCR and subsequently sequenced.
(The numbering, e.g. Q33, corresponds to a specific isolated clone; the sequences of the various scFv are aligned to each other; the complete sequence of each clone must be read continuously throughout the different blocks) g No.1:Q33 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIQRHGTWTGY
No.2:Q1.3 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSINYNGDATSY
No.3:Q12 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINAAGAQTGY
No.4:Q4 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIGQKGNKTTY
No.5:R2 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRITQSGTYTQY
No.6:Q15 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSINAFGQSTRY
No.7:R10 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINASGTLTRY
No.8:Q5 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISDTGSATTY
No.9:N6 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNISDAGCATYY
No.l0:Q32 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIHSAGQETIY
No.11:R6 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYITTNGSTTSY
No.12:Q9.3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYITTNGSTTSY
No.13:Q24 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITTSGGDTAY
No.14:Q3.1 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYINASGASTSY
No.15:Q25 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTITSSGQQTFY
No.16:N2 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSQGPVTWY
No.17:Q3.3 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISTSGSYTTY
No.18:Q21 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINGLGTPTAY
No.19:N4 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIQTSGRDTTY
No.20:R3 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAITQYGGDTGY
No.21:Q2 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISNLGQPTHY
No.22:Q30 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISNLGQLTHY
No.23:Q16 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIDPMGQSTNY
No.24:R5 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAITNTGQWTTY
No.25:Q26 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIQSVGTYTVY
No.26:Q34 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIPATGQRTFY
No.27:Q6.1 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISRTGKVTDY
No.28:Q1.2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIEAGGGETTY
No.29:R4 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIRPQGHPTQY
No.30:N1 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIRPPGQTTQY
No.31:R7 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSQIQENGVTTTY
Q1.3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSSTFDYWGQGTLVTVSSGGGG
Q9.3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDYSDFDYRGQGTLVTVSSGGGG
Q3.1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNTSDFDYRGQGTLVTVSSGGGG
Q3.3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGTTFDYWGQGTLVTVSSGGGG
Q6.1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKKMTSFDYWGQGTLVTVSSGGGG
Q1.2 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKATTTFDYWGQGTLVTVSSGGGG
Q1.3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDGVTITCRASQSISSYLNWYQQKPGKAPKLLI
, CA 02375033 2001-11-27 Q9.3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q3.1 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q3.3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q6.1 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q1.2 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q1.3 YSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTSNSPATFGQGTKVEIKR
Q9.3 YSASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNGATPNTFGQGTKVEIKR
Q3.1 YSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSAPATFGQGTKVEIKR
Q3.3 YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR
Q6.1 YDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRQPGTFGQGTKVEIKR
Q1.2 YDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRPPVTFGQGTKVEIKR
The object of the invention is also amino acid sequences of mimicry peptides against the MUC1 conformation epitope as well as all DNA sequences that encode these amino acid sequences, DNA
and peptide and partial peptide sequences that can be derived from them and that have the same characteristics.
More particularly, this concerns the amino acid sequences of mimicry peptides with the following sequence nos. 32 to 47.
(The numbering, e.g. S1, corresponds to a specific isolated clone; the sequences of the various peptides are aligned to each other) .
No.32:S1 CEYYDVPMARC
No.33:S12 CDYPSRLIDLC
No.34:Rol CGLACERPCGWVC
No.35:Ro5 CLGGCERPCMYSC
No.36:Rol3 CRGRCGEWCSRPC
No.37:Ro6 CRGRCDQRCSRPC
No.38:Rol2 CPARCGVPCAMGC
No.39:V11 CIPHRHDGC
No.40:V4 CQPHRYDKSLPC
No.4l:V10 CTTRLLNEDGSC
No.42:U7 LHGPLWD
No.43:U10 LHGPLGM
No.44:U6 LHGPLWE
No.45:U7a LHGPLWDGAAGAETVES
No.46:UlOa LHGPLGMGPLGPKLLKV
No.47:U6a LHGPLWEGPLGPKLLKV
Antigens that are not, or not exclusively, proteins or peptides (e. g. carbohydrate antigens) are, similar to conformation epitopes of proteins, perceived by the immune system as three-dimensional patterns of charges and other molecular interactions and, like them, are subject to limitations in the generation of a cellular immune response. In these cases, too, the selection of mimicry peptides by the phage display method according to the invention can result in the antigen being "rewritten" into a peptide sequence, thus permitting the DNA vaccination technique, see Example 2.
The object of the invention is also protein sequences of antiidiotypical antibody fragments against TF as well as amino acid sequences of mimicry peptides against the TF carbohydrate epitope, all DNA sequences that encode these amino acid sequences, and DNA and protein or peptide and protein and peptide partial sequences that can be derived from them and that have the same characteristics.
More particularly, this concerns the following protein sequences of antiidiotypical antibody fragments against TF with the following sequence nos. 48 to 71.
No. 48 - >H16 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSMIDGSGSQTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSDLDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SYSTPNTFGQGTKVEIKR
No. 49 - >P3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISYSGATTNYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSDASFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
DYGGPTTFGQGTKVEIKR
No. 50 -'>P8 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISATGGSTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAVDTAVYYCAKSSDGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
ASSAPATFGQGTKVEIKR
No. 51 - >H6 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISAQGLTTTYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGRSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RKLLPWTFGQGTKVEIKR
No. 52 - >H1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITELGRSTQYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKPWPHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
AARRPTTFGQGTKVEIKR
, CA 02375033 2001-11-27 No. 53 - >H13 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSKISELGRNTSYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKDITAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SMRMPPTFGQGTKVEIKR
No. 54 - >K3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIQWSGESTWYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSTSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RRHTPTTFGQGTKVEIKR
No. 55 - >K3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIQWSGESTWYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSTSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RRHTPTTFGQGTKVEIKR
No. 56 - >K4 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIQFSGQGTRYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKTLSTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQITQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
GYRQPTTFGQGTKVEIKR
No. 57 - >K2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIRPLGSATQYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSNMAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
TTRPPTTFGQGTKVEIKR
No. 58 - >J6 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDISEQGARTMYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSTPAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
MNNKPNTFGQGTKVEIKR
, CA 02375033 2001-11-27 IS
No. 59 - >E3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSQITGLGSQTRYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGETAFDYWGQGTLVTVSSGGGGSGDIQMTQSPSSLSASVGDRVTITCRAS
QSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRQQRPSTFGQ
GTKVEIKR
No. 60 - >K1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNITQMGMTTAYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGEQTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RRTHPQTFGQGTKVEIKR
No. 61 - >E5 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISQTGTRTKYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGSASFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPTRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
VTTHPNTFGQGTKVEIKR
No. 62 - >K2+
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCARQVKSWTRWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVALGQT
VRITCRGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRD
SSGNHYVFGGGTKLTVLG
No. 63 - >K4+
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMDSLRAEDTAVYYCARGRRKQDKSTRWGQGTLVTVSSGEGGSGGGGSGGSALSSELTQDPAVSVAL
GQTVRITCQGSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNS
RDSSGSSSVFGGGTKLTVLG
No. 64 - >K4-EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMDSLRAEDTAVYYCARGRRKQDKSTRWGQGTLVTVSGSGGGGSGGSALSSELTQDPAVSVALGQTV
RITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDS
SGSSSVFGGGTKLTVLG
, CA 02375033 2001-11-27 No. 65 - >K9+
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDN
AKNSLYLQMNSLRAEDTAVYYCARDPFHPWGQGTLVTVSSGGGGSGGGGSGGSALSSELIQDPAVSVALGQTVR
ITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSS
GTVFGGGTKLTVLG
No. 66 - >K1+
QVQLQESGPGLVKPSETLSLTCWSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPNYSPSLKSRATISVDK
SKNQFSLKLSSVTAADTAVYYCARQDMTQQTSWGQGTLVTVSSGGGGSGGGGSGGSALQSVLTQPPSASGTPGQ
RVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCA
AWDDSLRNLVFGEGTKLTVLG
No. 67 - >K3+
QVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPNYSPSLKSRATISVDK
SKNQFSLKLSSVTAADTAVYYCARQDMTQQTSWGQGTLVTVSSGEGGSGEGGSGGSALQSVLTQPPSASGTPGQ
RVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCA
AWDDSLRNLVFGEGTKLTVL
No. 68 - >ZA4 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQP
PGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDDK
GGWGQGTLVTVSSGGGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNIGSN
TVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAW
DDSLRSLVFGGGTKLTVLG
No. 69 - >ZA36 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYH
SGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARPSSIWGQGTLVTVSSG
GGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPK
LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRSLVFGGGTK
LTVLG
, CA 02375033 2001-11-27 No. 70 - >ZA14 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHS
GSTNYNPSLKSRVTISVXKSKNQFSLKLSSVTAXDTAVYYCARPSHHAGTHTWGQGTLVT
VSSGGGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPG
TAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLRALVFG
GGTKLTVLG
No. 71 - >Z9 QVQLQESGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGS
TNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARKGLNFGPWGQGTLVTVSSG
GGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNVGSNTVNWYQQLPGTAPK
LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLRSYVFGGGTK
LTVLG
Furthermore, this concerns the amino acid sequences of mimicry peptides against the TF carbohydrate epitope with the following sequence nos. 72 to 96.
(The numbering, e.g. S1, corresponds to a specific isolated clone) No.72:T1 CLREGHFASFC
No.73:T14 CGMLTPAWIKC
No.74:T4 CETFSNLAFLC
No.75:T7 CEGPEIPAFVC
No.76:T3 CESMVEPAWVC
No.77:T15 CTNDIMPPWVC
No.78:T2 CDGLLLPIWAC
No.79:T11 CAGEFVPVWAC
No.80:T16 CDLGLKPAWLC
No.8l:X3 CGPMCSGSCVPQC
No.82:X9 CDAGCNFFCPWRC
No.83:X2 CGPMCSGSCXPQC
Ig No.84:Y8 VWWWQWS
No.85:Y1 MWRPFWL
No.86:Y4 PPWVXHL
No.87:Y9 LIPQWIV
No.88:W4 CTPADMSGC
No.89:W3 CTPADMSGC
No.90:W16 CPSVWMLDLGPC
No.9l:W15 CHGGLTPLC
No.92:W8 CGPMMLWHW
No.93:W5 CTRHIHWGNAHW
No.94:W14 CTPADMSGW
No.95:A1 CFRGGPWWSLC
No.96:A2 CAVRTWVISEC
The invention is explained in more detail by examples of embodiments, however, it is not limited to these.
Examples of embodiments Example 1 Production of hybridome cell line A76-A/C7 and of antibodies After treatment with Neuraminidase (V. cholerae), Balb/c mice were immunized i.p. with a suspension of live human mammacarcinoma cells of the cell line T-47D (Keydar, I. et al., Eur J Cancer, 15:659, 1979) without an adjuvant. The fusion cell line was X63-Ag8.653 (Kearney, J.F. et al., J Immunol 123:1548, 1979). The hybridome method itself was carried out according to standard methods (e. g. Peters, H.H., et al., "Monoklonale Antikorper, Herstellung and Charakterisierung" [Monoclonal Antibodies, Production and Characterization], Berlin 1985;
Friemel, H. "Immunologische Arbeitsmethoden" [Immunological Working Methods], 4th edition, Jena 1991). The specificity analysis of the monoclonal antibodies (mAK) produced by the hybridome cell lines was based on enzyme immunoassays with natural glycoproteins and synthetic peptides and glycopeptides, immunofluorescence analyses with diverse cell lines as well as immune histochemical studies of tissue sections. For the monoclonal antibody A76-A/C7, the epithelial mucin, MUC1, was unequivocally determined as the specific antigen. IgGl,k was determined as the isotope, with a small portion of IgM of the same specificity using a commercial isotyping kit (Pharmingen, San Diego, USA). An epitope mapping within the framework of the ISOBM TD-4 International Workshop on Monoclonal Antibodies against MUC1 (Tumor Biol. 19, Suppl. 1, 1998) defined the epitope as APDTRPAP. Additional studies using synthetic, glycosylated and non-glycosylated peptides showed that the epitope of the monoclonal antibody A76-A/C7 is, to great extent, determined by its conformation:
- The antibody binds only insignificantly to a single unit (a repeat), despite its containing the epitope sequence.
- The binding to non-glycosylated peptides depends on the length of the peptide or, more specifically, on the number of linked repeats (Fig. la). It is known from the literature that the native conformation of the PDTRP
motive only develops with a peptide length of more than 3 repeats (Fontenot, J.D., et al., J Biomol Struct Dyn 13:245, 1995).
- The binding of the monoclonal antibody A76-A/C7 to a single MUCl unit (1 repeat) is greatly increased if it is glycosylated with GalNAc-3GalNAc or Galal-3GalNAc in the area of the epitope at the Thr (Fig. lb~ see also Karsten, U., et al., Cancer res, 58:2541, 1998).
The antibody was cleaned by ammonium sulphate precipitation followed by an affinity chromatography on protein A-Sepharose.
Obtaining human recombinant antibody fragments that mimic the conformation-dependent epitope of the MUC1 from antibody gene libraries using the phage display method Two different synthetic antibody gene libraries were used which represent human single-chain antibody fragments (scFv). One antibody gene library (Griffin 1 Library; http://www.mrc-cpe.cam.ac.uk/-phage/) comprises more than 109 phages with various combinations of the variable regions of the heavy and light chains of human antibodies with, in part, randomized hypervariable regions that are connected by a peptide piece (linker) and are covalently bound to a phage coat protein (pIII). It is derived from a different antibody gene library (Griffiths, A. et al., 1994, EMBO I., 13:3245-3260). The second, smaller gene library comprises scFv with the same framework (single framework library) which were preselected for active folding of the antibody fragments by binding to protein L and protein A (I. Tomlinson., 9th anniversary conference: "Antibody engineering", IBC Conferences, San Diego 1998; I. Tomlinson, 10th anniversary conference: "Antibody engineering" IBC
Conferences, San Diego 1999; Speaker abstract). The first library originates from the Dr. G. Winter Laboratory and the second from the Dr. I. Tomlinson Laboratory (both MRC Centre for Protein Engineering, Cambridge, U.K.). The specific phages were selected in 2-3 rounds (phage panning) using the proteolytic selection method with the helper phage KM13 (Kristensen, P. and Winter, G. Folding & Design, 3:321, 1998). The cleaned monoclonal antibody A76-A/C7 (35 u/ml in 4 ml) was used as the antigen which was immobilized overnight in a test tube (Immunotube, Nunc, Wiesbaden) in PBS at 4 degrees Celsius.
Alternatively, A76-A/C7 was incubated with the phages; the phages bound to the antibodies were obtained through magnet beads with immobilized anti-IgG antibodies (Deutsche Dynal, Hamburg) . After stringent washing steps (up to 20 times PBS/
O.lo Tween20 followed by 20 times PBS), the phages specifically bound during the selection rounds (3 h at RT) were eluted through the tandem repeat (100 ug/ ml; Biosynthan, Berlin-Buch) that was glycosylated with GalNAc in the PDTR, and subsequently treated with Trypsin (proteolytic selection method). Between the selection rounds, the eluted phages were multiplied with helper phages in the bacteria and selected once again.
Obtaining mimicry peptides that mimic the conformation-dependent epitope of the MUCI from peptide gene libraries using the page display method Analogous to the example for generating antiidiotypical antibodies, in multiple selection rounds specifically binding peptides were obtained from a peptide gene library (gene library of Dr. H. Gollasch; Oligino, L. et al., J Biol Chem 272:29046, 1997) that has 107 different short peptides coupled to the phage coat protein pIII. The expressed peptides are randomized nonapeptides that are flanked , and thus circularized, by two cysteins (CX9C), thus increasing the stability and the affinity.
The selection and testing were carried out as described in the generation of the antiidiotypical antibodies. Similarly, additional linear and circular mimicry peptides were obtained with other peptide libraries. These are peptide libraries that were created in the same way as the aforementioned peptide library. The expressed peptides are linear peptides with 7 amino acids and circular peptides with 7 randomized amino acids flanked by two cysteins (CX10C), circular peptides with 10 randomized amino acids flanked by two cysteins (CX10C), and circular peptides with a total of 9 randomized amino acids with two internal and two flanking cysteins (CX3CX3CX3C).
Specificity tests of the mimicry peptides and the antiidiotypical antibody fragments The selected peptides and antibody fragments were tested for their binding to the monoclonal antibody A76-A/C7 in ELISA tests as well as in the form of a negative control with other IgG and IgM monoclonal antibodies. Furthermore, in ELISA tests they were tested for their binding to a series of well characterized MUC1-specific antibodies that differ in their fine specificity.
For the ELISA tests, the form of peptides and antibody fragments coupled to phages was used. The antiidiotypical svFr and the mimicry peptides can be categorized in groups that:
- bind exclusively to A76-A/C7;
- bind to A76-A/C7 and other MUC1-specific antibodies that bind only to either the conformation epitope (in the PDTR region glycosylated MUC1 tandem repeat) (type A) or whose binding is greatly increased through the PDTR
glycosylation of the MUC1 tandem repeat (conformation induction) (type B) - bind to MUCl-specific antibodies that, besides type A and type B, also bind MUCl-specific antibodies which bind glycosylated and unglycosylated MUC1 tandem repeats to the same extent (type D) - bind strongly to MUC1-specific antibodies and that, with regard to the glycosylation of the PDTR region of the MUC1 repeat to A76-A/C7, behave quite the opposite and do not bind the glycosylated MUC1 peptide or bind much less than the non-glycosylated MUC1 peptide (type C). These mimicry peptides or antiidiotypical scFv can also bind to other types of the MUC1-specific antibody.
The mimicry peptides and antiidiotypical antibody fragments were also tested in ELISA inhibition tests to see whether they, in the form of the syntheticized peptides or cleaned scFv (alone or coupled to phages), specifically and concentration-dependently inhibit the binding of A76-A/C7 to the glycosylated MUC1 peptide (in the epitope PDTR with GalNAc glycosylated tandem repeat) and non-glycosylated oligomers of the 20-mer tandem repeats. These tests were carried out with streptavidin-coated micro test plates (BioTeZ, Berlin-Buch) and biotinylated MUC1 peptides (Biosynthan, Berlin-Buch; Fig. lc) as well as with normal ELISA test plates on which the MUC1 peptides were immobilized by drying.
Inbred mice of the Balb/c line were immunized intraperitoneally with mimicry peptides and antiidiotypical antibody fragments in the form of the synthesized peptides or cleaned scFv alone, respectively coupled to the protein KLH or to bacteriophages in PBS, mixed with an incomplete Freund's adjuvant, whereby mixtures of antiidiotypical scFv phages or mimicry peptide phages from the respective groups (see above) were used. Three weeks later a booster of the same preparation, but without the adjuvant, was administered. The booster was repeated after three weeks and ten days later blood samples were taken from the mice. In ELISA tests the serum was tested for antibodies that specifically recognize the conformation-dependent epitope of the MUC1 (test setup as above). The compositions of the antiidiotypical scFv as well as the mimicry peptides trigger a strong reaction against the conformation-dependent epitope of the MUCl.
Design of the DNA vaccines and testing on mice The antiidiotypical scFv were directionally cloned into a DNA
vaccination vector, whereby the scFv were cut out of the phage vector by means of Sfil and NotI and directionally cloned into various DNA vaccination vectors that had previously been split with the same enzymes. A suitable vector for this is the vector pVAC2 (I. Harmer et al., Keystone Symposium "DNA Vaccines", Snowbird, USA, 1999; poster and poster abstract) which, after insertion of the scFv into the DNA vaccination vector, encodes a fusion protein from the antiidiotypical scFv with a tetanus toxoid. The tetanus toxoid has the characteristic of an adjuvant and strengthens the immune response against the fused protein portion (C. King et al., 1998, Nat Medicine 4:1281-86).
The mimicry peptides were also cloned into various DNA vaccine vectors. The cloning occurred according to the well known method of PCR cloning in which, using synthetic primers, the sequences that encode for the mimicry peptides were inserted into the DNA
vaccination vectors. Thus DNA vaccination vectors were also produced on the basis of the pVAC2 which were encoded with the tetanus toxoid for a fusion protein of the mimicry peptide.
The DNA of the vaccination vectors was increased according to well known methods, cleaned and then injected into mice.
Mixtures of DNA vaccination vectors that encode antiidiotypical scFv or mimicry peptides as the fusion protein using the tetanus toxoid and which respectively originate from the various groups with different binding patterns for MUC1-specific antibodies (see above) were used for the immunization. The dose was 50 ug or 200 ug, respectively, of total DNA and administration was intramuscular. Four weeks later a booster of the same preparation was administered and then repeated again after 4 weeks. 10 days later blood samples were taken from the mice. In ELISA tests the serum was tested for antibodies that specifically recognize the conformation-dependent epitope of the MUC1 (test setup as above).
The immunization with the mixtures of DNA vaccine vectors showed a strong humoral immune response against the conformation-dependent epitope of the MUCl and a strong response against the tetanus toxoid in the antiidiotypical scFv as well as the mimicry peptides of the coding DNA vectors.
Vaccines in the tumour challenge model In the mouse tumour challenge model, different mouse tumour cells (3T3 and P815) were used that stably transfect with the cDNA of the transmembrane form of the human MUC1. The MUC1-positive mouse cell lines express the conformation epitope of the MUC1 which was tested in immune binding studies with the A76-A/C7. Several mouse lines were used for the studies (Balb/c, DBA/2 and C57BL/6). After vaccinating the mice according to the prime boost protocol described below, the mice were subcutaneously injected near the peritoneum with 106 to 10' tumour cells in 200 a PBS and the tumour growth (tumour size in mm) was measured over 20 - 30 days.
Prime boost vaccination pattern:
For the immunizations (priming), a combination of DNA
vaccination vectors (encoding for scFv tetanus toxoid or mimicry peptide tetanus toxoid fusion protein) was used with two candidates from each of the 4 different groups of antiidiotypical scFv or mimicry peptides. However, for the booster, the same combinations of antiidiotypical scFv or mimicry peptides were used in incomplete Freund's adjuvants, but in their protein form. To this end, the scFv were cleaned by nickel chelate chromatography according to well known procedures and the mimicry peptides were coupled to KLH according to well known procedures. For the immunization, 50 - 200 ug of total DNA
were administered intramuscularly and for the scFv and the mimicry peptides, 10 - 200 ug were administered intraperitoneally at three week intervals and boosters were administered 2 - 3 times.
As a control, the DNA vaccination vectors were used for a scFv with a specificity against an irrelevant bacterial protein or for an irrelevant peptide (SSGSSSSGS), or their cleaned scFv or the peptide KLH complex. 5 - 10 animals were studied for the different test preparations.
The test showed that a vaccination according to the prime boost protocol prevents the growth of injected MUCl-positive mouse tumour cells or reduces them to a minimal size (0 - 20 mm2 after 20 days). In a subsequent injection with the same tumour cells without transfected MUC1, the same vaccination achieves an average tumour size of over 200 mm2 (after 20 days). The injection of MUC1-positive mouse tumour cell lines into mice without prior vaccination results in strong tumour growth (>200 mm2 after 20 days). An immunization and booster with the proteins of the antiidiotypical scFv or the mimicry peptides coupled to KLH without DNA vaccination vectors results in an immune response against the MUC1 tumour cells, however, the tumour protection is much less than with the prime boost protocol with the DNA vaccination vectors.
The results show that a vaccination with DNA vaccination vectors that encode for antiidiotypical scFv or mimicry peptides provides excellent protection against tumours. This reaction is MUCl-specific. It is far better or even possible compared to vaccination studies with the proteins of the antiidiotypical scFv or mimicry peptides without prior immunization with the corresponding DNA vaccination vectors.
This shows that the vaccines against conformation-dependent antigens according to the invention using DNA vaccination vectors of mimicry structures is a successful form of fighting tumours that carry these conformation-dependent antigens.
Example 2 Production of hybridome cell lines A78-G/A7 and of antibodies In the case of A78-G/A7 (see also Karsten, U. et al., Hybridoma 14:37, 1995), Balb/c mice were immunized intraperitoneally with 100 ug of Asialoglycophorin (Sigma, Diesenhofen) in PBS mixed with Freund's adjuvant. After 24 h, 100 ug of cyclophosphamid in PBS per kg body weight was administered i.p. A booster of 100 ug Asialoglycophorin was administered two weeks later. Each time the fusion cell line was X63-Ag8.653 (Kearney, J.F. et al., J
Immunol 123:1548, 1979). The hybridome method was carried out according to standard methods (e. g. Peter, H.H. et al., "Monoklonale Antikorper, Herstellung and Charakterisierung"
[Monoclonal Antibodies, Production and Characterization], Berlin 1985; Friemel, H. "Immunologische Arbeitsmethoden"
[Immunological Working Methods], 4th edition, Jena 1991). The specificity analysis of the monoclonal antibodies produced by the hybridome cell lines was based on enzyme immunoassays with natural glycoproteins, synthetic peptides and glycopeptides, glycolipids and neoglycolipids and synthetic polyacrylamide-carbohydrate conjugates, absorption analyses of synthetic carbohydrate conjugates (Synsorb, Chembiomed, Edmonton, Canada), immunofluorescence analyses with diverse cell lines as well as immune histochemical studies of tissue sections. For the A78-G/A7, the carbohydrate epitope Thomsen-Friedenreich (TF) that is associated with tumours was unequivocally determined as a specific antigen:
- A78-G/A7 binds exclusively to the disaccharide TF in the a-anomer configuration (TFa; Gall-3GalNAca1-0-Ser/Thr) on natural and synthetic structures as occur naturally only on glycoproteins in the form of a direct 0-glycoside binding to serines or threonines. However TFa, which can occur at the end of glycan chains of glycolipids, as well as other carbohydrate structures, portions of peptides or lipids, are not bound.
- A78-G/A7 binds highly specifically to various cancer cell lines in immunofluorescence studies and to various cancers in histochemical studies. (Cao, Y. et al., Histochem Cell Biol 106:197, 1996; Cao, Y. et al., Cancer 76:1700, 1995; Cao, Y.
et al., Virchows Arch 431:159, 1997; Karsten, U. et al., Hybridoma 14:37, 1995.
- For A78-G/A7, IgM, k, was determined as the isotype using a commercial isotyping kit (Pharmingen, San Diego, USA).
A78-G/A7 was cleaned using an ammonium sulphate precipitation followed by an affinity chromatography on a proteinG affinity matrix for cleaning undesirable IgG antibodies from the calf serum and, finally, with an affinity chromatography using a goat-anti-mouse-Ig affinity matrix (Perzellulose, BioTeZ, Berlin-Buch) (Dr. G. Butschak).
Production of human recombinant antibody fragments against the Thomsen-Friedenreich antigen from antibody libraries using the phage display method Two different synthetic antibody gene libraries were used which represent human single-chain antibody fragments (scFv). One antibody gene library comprises more than 101° phages with various combinations of the variable regions of the heavy and light chains of human antibodies with, in part, randomized hypervariable regions that are connected by a peptide piece (linker) and are covalently bound to a phage coat protein (pIII). It is derived from a different antibody gene library (Griffiths, A. et al., 1994, EMBO I., 13:3245-3260). The second, smaller gene library comprises scFv which were preselected for active folding of the antibody fragments. The first library originates from the Dr. G. Winter Laboratory and the second from the Dr. I. Tomlinson Laboratory (both MRC Centre for Protein Engineering, Cambridge, U.K.). The specific phages were selected in 2-3 rounds (phage panning) using the proteolytic selection method with the helper phage KM13 (Kristensen, P. and Winter, G. Folding and Design, 3:321, 1998). The cleaned monoclonal antibody A78-G/A7 (35 u/ml in 4 ml) was used as the antigen which was immobilized overnight in a test tube (Immunotube, Nunc, Wiesbaden) in PBS at 4 degrees Celsius.
Alternatively, the cleaned antibody was incubated with the phages; the phages bound to the antibodies were obtained through magnet beads with immobilized anti-IgM antibodies (Deutsche Dynal, Hamburg). After stringent washing steps (up to 20 times PBS/ O.lo Tween20 followed by 20 times PBS), the phages specifically bound during the selection rounds (3 h at RT) were specifically eluted through the TFa-carrying glycoprotein asialglycophorin (100 - 165 ug/ ml) and, in part, subsequently treated with Trypsin (proteolytic selection method). Between the selection rounds, the eluted phages were multiplied with helper phages in the bacteria and selected once again. 2 to 3 selection rounds were carried out.
Identification of peptides using a peptide gene library that specifical3y mimics the Thomsen-Friedenreich antigen Analogous to the example for generating antiidiotypical antibodies, in multiple selection rounds specifically binding peptides were obtained from a peptide gene library (Oligino, L.
et al., J Biol Chem 272:29046, 1997) that has 10' different short peptides coupled to the phage coat protein pIII (in cooperation with Dr. H. Gollasch Robert-Rossle-Klinik, Berlin-Buch). The expressed peptides are randomized nonapeptides that are flanked, and thus circularized, thereby increasing the stability and the affinity. The selection and testing were carried out as described in the generation of the antiidiotypical antibodies.
Specificity testing of the mimicry peptides and antiidiotypical antibody fragments The selected peptides and antibody fragments were tested in ELISA tests for their binding to the TF-specific antibodies and to the plant lectin PNA (peanut agglutinin, Arachis hypogaea lectin; Sigma) which also , if not exclusively, binds the Thomsen-Friedenreich antigen, as well as for comparison with other IgM and IgG antibodies. To this end, the form of peptides and antibody fragments that are coupled to phages were used which were cleaned beforehand through a polyethylene glycol precipitation in 96-well plates. The potential mimicry peptides and antiidiotypical antibody fragments were then studied in ELISA inhibition tests as to whether they specifically inhibit the binding of A78-G/A7 and/or other TF-recognizing antibodies and lectins to the disaccharide TFa. To this end, the TFa-carrying glycoprotein asialglycophorin was immobilized on ELISA
plates by drying, and the binding of the monoclonal antibodies and lectins was concentration-dependently inhibited by the mimicry peptides or antiidiotypical antibody fragments in the form of the synthetic peptides or cleaned scFv alone or coupled to phages (Fig. 2).
~
Inbred mice of the Balb/c and NMRI lines were immunized intraperitoneally with mimicry peptides and antiidiotypical antibody fragments in the form of the synthesized peptides or cleaned scFv alone, respectively coupled to the protein KLH or to bacteriophages in PBS, mixed with a complete Freund's adjuvant. Three weeks later a booster of the same preparation, but without the adjuvant, was administered. The booster was repeated after three weeks and ten days later blood samples were taken taken from the mice. In ELISA tests the serum was tested for antibody bindings against the Thomsen-Friedenreich antigen.
Vaccination with TF-mimicking peptides in the mouse tumour model Cell culture: The mouse colon cancer cell line C-26 was kept in the medium RPMI 1640 with the addition of loo foetal calf serum.
Tumour model: 105 cells of the syngene colon cancer cell line C-26 were transplanted subcutaneously into mice of the Balb/c line in two variants: a) untreated and b) pretreated (TF-positive) with neuraminidase from V. cholerae (Serva, Heidelberg). At weekly intervals the tumour size was determined externally.
After 3 weeks the animals were killed and the livers removed in order to determine the number of visible metastases on the surface of the liver.
Vaccination: The vaccination of the mice was begun 6 weeks prior to the tumour transplant. The phage preparation or the cleaned scFv (as well as corresponding controls) were emulgated 1:1 with an incomplete Freund adj uvants and inj ected i . p . Four weeks later a booster was administered (without adjuvant). After two more weeks, the tumour transplant (tumour challenge) was carried out with untreated and C-26 cells treated with neuraminidase.
Results: The present results with three of the aforementioned antiidiotypical scFv showed that the initial rate of tumours in the C-26 cells treated with neuraminidase can be significantly reduced through vaccination (to 3 - 16% of the control; control:
1000 initial rate). Furthermore, the number of liver metastases in the vaccinated animals approximately corresponded to that of the animals that were transplanted with untreated (TF-negative) C-26 cells (ca. 2 per liver), whereas the unvaccinated control animals with TF-positive C-26 cells had 5 - 9 metastases per liver.
Legends for the Figures:
Fig. lc:
Inhibition of the A76-A/C7 binding to the MUC1 glycopeptide (Biotin-Ahx-APPAHGVTSAPD-Thr(a-D-GalNAc)-RPAPGSTAPPAHGVTSA) through scFv phages. The MUC1 glycopeptide was immobilised on the streptavidin ELISA plate (5 ng/well) and subsequently blocked with 30% FKS in RPMI. The culture supernatant of the A76-A/C7 (diluted 1:80 ) was preincubated for one hour with the scFv phages that were cleaned through a polyethylene glycol precipitation in the indicated concentrations (percent by volume of adjusted phage solutions in PBS) and subsequently placed on the MUC1 glycopeptide plate for 2 hours. Verification occurred via an anti-mouse-POD antibody (Dako). The scFv phages Q6, Q7 and Q8 are examples of antiidiotypical scFv, whereas Q4 and Q10 are examples of control scFv that bind the A78-A/C7 but are not, however, antiidiotypical scFv.
Figure 2:
Inhibition of the A78-G/A7 binding to Asialoglycophorin through scFv phages. The Asialoglycophorin (A-GP) was immobilized on the ELISA plate by drying (25 ng/well) and subsequently blocked with 30% FKS in RPMI. The culture supernatant of the A78-G/A7 (diluted 1:20 ) was preincubated for one hour with the scFv phages that were cleaned through a polyethylene glycol precipitation in the indicated concentrations (percent by volume of adjusted phage solutions in PBS) and subsequently placed on the A-GP plate for 2 hours. Verification occurred via an anti-mouse-POD antibody (Dako). The scFv phages P9, P13, P16, P3 and K3 are examples of antiidiotypical scFv, whereas P8 and Ql are examples of control scFv, of which P8 binds the A78-G/A7 but is not, however, an antiidiotypical scFv, and Ql is a phage that does not bind A78-G/A7.
Alternatively, antiidiotypical antibodies are obtained by means of the hybridome method and fragments are isolated from them, if required. These mimicry peptides are rewritten into a DNA
and used as a DNA vaccine. One process is what is known as the prime boost protocol, in which the intradermal, intramuscular or intrarectal injection of a DNA (priming) in the form of a plasmid DNA, linear DNA or a plasmid replicon vector is followed by a booster with the corresponding antigen, alone, in the form of a chemical coupling of proteins, in the form of bacteriophages as fusion proteins with phage coat proteins on their surface, in the form of a fusion protein on the surface of other viruses or attenuated biological carriers or in the form of dendritic cells loaded with a peptide. In this case, the DNA as well as the expressed mimicry peptide are required, which is easy using the phage display or ribosome display method. Alternatively, a corresponding recombinant virus-vector particle (e. g. fowlpox, constructs derived from adeno or alpha virus) can be used successfully. The immune response can be significantly strengthened through the additional administration of suitable cytokines, also in the form of a DNA, through immunostimulating CpG-DNA motives (non-methylated cytosine-guanine dinucleotides) or through suitable adjuvants (e. g.
aluminium phosphates).
Besides vaccines against conformation-dependent antigens, the invention also refers to vaccines against antigens that are not, or not exclusively, proteins or peptides according to Claim 3.
A target antigen type of the group of antigens that are not, or not exclusively, proteins or peptides are glycostructures;
additional immunogenic structures are combined carbohydrate-protein epitopes, lipids, glycolipids or synthetic structures.
A process is known from DE 196 27 352 A1 with which a monoclonal antiidiotypical antibody can be obtained using the hybridome method, which immunologically mimics pure carbohydrate structures. According to the invention, starting with this antiidiotypical antibody, a vaccine (preferably a DNA vaccine of this antibody or a suitable fragment thereof) is used for the vaccination. Thus, the present invention expands several points of this process from DE 196 27 352 A1. Antiidiotypical antibody fragments can be obtained directly from the antibody gene libraries using the phage display method or the ribosome display method. Also with this process, human antibody fragments can be obtained directly. In addition, combined carbohydrate-peptide epitopes can also be used. Plus there is a process with which short linear or circular peptides which immunologically mimic the antigen (also known as mimicry peptides) can be obtained from peptide gene libraries, also using the phage display method or the ribosome display method. To this end, not only specific idiotypical antibodies (Abl) are used as primary reagents for the selection of these mimicking structures, but also other substances that specifically recognize the glycostructure, such as lectins or receptors. The process also includes the use of the obtained structures preferably as DNA
vaccines, alone or in conjunction with the antibodies that immunologically mimic the antigen, antibody fragments or peptides in a suitable formulation (see above and claims), for example in a suitable formulation of the prime boost protocol.
Furthermore, according to the invention the mimicking protein structures can also be used alone for vaccination.
The invention also refers to vaccines (in the full scope of the description for conformation-dependent antigens) against the antigens glycopeptides, glycolipids, lipids, synthetic structures or other antigens that are not, or are only partially, proteins or peptides, the relevant epitopes having improved immunological structures, as well as to their production processes and their use.
The immunotherapy approach to diseases involving tumours is based on the assumption that it is possible to strengthen or activate the natural immune response. The rationale for vaccination lies in combating the residual disease (metastasis prophylaxis) according to a conventional therapy (e. g. surgical removal of the main body of tumour cells). As the name implies, mimicry peptides immunologically mimic the original antigen or epitope. They do this to very high degree, but not completely.
This can be seen as positive for applications within the framework of a vaccine (and more particularly in the case of a tumour vaccine) in that specifically inhibiting processes, e.g.
tolerance phenomena, are circumvented.
The prerequisite for the development of defined tumour vaccines is not only the presence of tumour-specific antigens, but also knowledge thereof. Great progress has been achieved in this area during the past three decades, not least through the development of monoclonal antibodies.
One widespread cancer antigen is the epithelial mucin, MUC1, whose immune-dominant epitope occurs multiple times on the extracellular part of the molecule. In its native state, this epitope forms a type I-(3 turn, but on synthetic peptides only under certain conditions, e.g. when the theonin of the dominant immune region is glycosylated with GalNAca1-0-Thr or Gal~il-3GalNAca1-0-Thr (Karsten, U. et al., Cancer res 58:2541-2549, 1998). As a rule, this epitope is perceived as a typical conformation epitope by the immune system, see Example 1.
According to the invention, using the phage display method, this conformation epitope is mimicked by immunologically identical (or almost identical) sequential epitopes which, in the form of a DNA, are part of a tumour vaccine in a DNA vaccination vector (Example 1) .
Therefore, the object of the invention is also human antiidiotypical antibody fragments against the MUC1 conformation epitope as well as all DNA sequences that encode these fragments, and protein sequences or DNA or protein partial sequences that can be derived from them and that have the corresponding characteristics.
Primarily, this concerns the following human antiidiotypical antibody fragments against the MUC1 conformation epitope with the following sequence nos. 1 to 31.
Fragments that contain the desired DNA of the scFv and of the peptides were multiplied using the PCR and subsequently sequenced.
(The numbering, e.g. Q33, corresponds to a specific isolated clone; the sequences of the various scFv are aligned to each other; the complete sequence of each clone must be read continuously throughout the different blocks) g No.1:Q33 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIQRHGTWTGY
No.2:Q1.3 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSINYNGDATSY
No.3:Q12 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINAAGAQTGY
No.4:Q4 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIGQKGNKTTY
No.5:R2 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRITQSGTYTQY
No.6:Q15 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSINAFGQSTRY
No.7:R10 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINASGTLTRY
No.8:Q5 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISDTGSATTY
No.9:N6 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNISDAGCATYY
No.l0:Q32 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIHSAGQETIY
No.11:R6 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYITTNGSTTSY
No.12:Q9.3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYITTNGSTTSY
No.13:Q24 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITTSGGDTAY
No.14:Q3.1 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYINASGASTSY
No.15:Q25 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTITSSGQQTFY
No.16:N2 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSQGPVTWY
No.17:Q3.3 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISTSGSYTTY
No.18:Q21 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINGLGTPTAY
No.19:N4 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIQTSGRDTTY
No.20:R3 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAITQYGGDTGY
No.21:Q2 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISNLGQPTHY
No.22:Q30 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISNLGQLTHY
No.23:Q16 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIDPMGQSTNY
No.24:R5 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAITNTGQWTTY
No.25:Q26 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIQSVGTYTVY
No.26:Q34 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIPATGQRTFY
No.27:Q6.1 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISRTGKVTDY
No.28:Q1.2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIEAGGGETTY
No.29:R4 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIRPQGHPTQY
No.30:N1 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIRPPGQTTQY
No.31:R7 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSQIQENGVTTTY
Q1.3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSSTFDYWGQGTLVTVSSGGGG
Q9.3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDYSDFDYRGQGTLVTVSSGGGG
Q3.1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNTSDFDYRGQGTLVTVSSGGGG
Q3.3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGTTFDYWGQGTLVTVSSGGGG
Q6.1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKKMTSFDYWGQGTLVTVSSGGGG
Q1.2 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKATTTFDYWGQGTLVTVSSGGGG
Q1.3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDGVTITCRASQSISSYLNWYQQKPGKAPKLLI
, CA 02375033 2001-11-27 Q9.3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q3.1 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q3.3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q6.1 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q1.2 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q1.3 YSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTSNSPATFGQGTKVEIKR
Q9.3 YSASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNGATPNTFGQGTKVEIKR
Q3.1 YSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSAPATFGQGTKVEIKR
Q3.3 YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR
Q6.1 YDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRQPGTFGQGTKVEIKR
Q1.2 YDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRPPVTFGQGTKVEIKR
The object of the invention is also amino acid sequences of mimicry peptides against the MUC1 conformation epitope as well as all DNA sequences that encode these amino acid sequences, DNA
and peptide and partial peptide sequences that can be derived from them and that have the same characteristics.
More particularly, this concerns the amino acid sequences of mimicry peptides with the following sequence nos. 32 to 47.
(The numbering, e.g. S1, corresponds to a specific isolated clone; the sequences of the various peptides are aligned to each other) .
No.32:S1 CEYYDVPMARC
No.33:S12 CDYPSRLIDLC
No.34:Rol CGLACERPCGWVC
No.35:Ro5 CLGGCERPCMYSC
No.36:Rol3 CRGRCGEWCSRPC
No.37:Ro6 CRGRCDQRCSRPC
No.38:Rol2 CPARCGVPCAMGC
No.39:V11 CIPHRHDGC
No.40:V4 CQPHRYDKSLPC
No.4l:V10 CTTRLLNEDGSC
No.42:U7 LHGPLWD
No.43:U10 LHGPLGM
No.44:U6 LHGPLWE
No.45:U7a LHGPLWDGAAGAETVES
No.46:UlOa LHGPLGMGPLGPKLLKV
No.47:U6a LHGPLWEGPLGPKLLKV
Antigens that are not, or not exclusively, proteins or peptides (e. g. carbohydrate antigens) are, similar to conformation epitopes of proteins, perceived by the immune system as three-dimensional patterns of charges and other molecular interactions and, like them, are subject to limitations in the generation of a cellular immune response. In these cases, too, the selection of mimicry peptides by the phage display method according to the invention can result in the antigen being "rewritten" into a peptide sequence, thus permitting the DNA vaccination technique, see Example 2.
The object of the invention is also protein sequences of antiidiotypical antibody fragments against TF as well as amino acid sequences of mimicry peptides against the TF carbohydrate epitope, all DNA sequences that encode these amino acid sequences, and DNA and protein or peptide and protein and peptide partial sequences that can be derived from them and that have the same characteristics.
More particularly, this concerns the following protein sequences of antiidiotypical antibody fragments against TF with the following sequence nos. 48 to 71.
No. 48 - >H16 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSMIDGSGSQTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSDLDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SYSTPNTFGQGTKVEIKR
No. 49 - >P3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISYSGATTNYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSDASFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
DYGGPTTFGQGTKVEIKR
No. 50 -'>P8 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISATGGSTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAVDTAVYYCAKSSDGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
ASSAPATFGQGTKVEIKR
No. 51 - >H6 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISAQGLTTTYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGRSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RKLLPWTFGQGTKVEIKR
No. 52 - >H1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITELGRSTQYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKPWPHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
AARRPTTFGQGTKVEIKR
, CA 02375033 2001-11-27 No. 53 - >H13 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSKISELGRNTSYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKDITAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SMRMPPTFGQGTKVEIKR
No. 54 - >K3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIQWSGESTWYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSTSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RRHTPTTFGQGTKVEIKR
No. 55 - >K3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIQWSGESTWYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSTSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RRHTPTTFGQGTKVEIKR
No. 56 - >K4 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIQFSGQGTRYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKTLSTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQITQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
GYRQPTTFGQGTKVEIKR
No. 57 - >K2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIRPLGSATQYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSNMAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
TTRPPTTFGQGTKVEIKR
No. 58 - >J6 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDISEQGARTMYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSTPAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
MNNKPNTFGQGTKVEIKR
, CA 02375033 2001-11-27 IS
No. 59 - >E3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSQITGLGSQTRYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGETAFDYWGQGTLVTVSSGGGGSGDIQMTQSPSSLSASVGDRVTITCRAS
QSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRQQRPSTFGQ
GTKVEIKR
No. 60 - >K1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNITQMGMTTAYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGEQTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RRTHPQTFGQGTKVEIKR
No. 61 - >E5 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISQTGTRTKYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGSASFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPTRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
VTTHPNTFGQGTKVEIKR
No. 62 - >K2+
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCARQVKSWTRWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVALGQT
VRITCRGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRD
SSGNHYVFGGGTKLTVLG
No. 63 - >K4+
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMDSLRAEDTAVYYCARGRRKQDKSTRWGQGTLVTVSSGEGGSGGGGSGGSALSSELTQDPAVSVAL
GQTVRITCQGSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNS
RDSSGSSSVFGGGTKLTVLG
No. 64 - >K4-EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMDSLRAEDTAVYYCARGRRKQDKSTRWGQGTLVTVSGSGGGGSGGSALSSELTQDPAVSVALGQTV
RITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDS
SGSSSVFGGGTKLTVLG
, CA 02375033 2001-11-27 No. 65 - >K9+
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDN
AKNSLYLQMNSLRAEDTAVYYCARDPFHPWGQGTLVTVSSGGGGSGGGGSGGSALSSELIQDPAVSVALGQTVR
ITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSS
GTVFGGGTKLTVLG
No. 66 - >K1+
QVQLQESGPGLVKPSETLSLTCWSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPNYSPSLKSRATISVDK
SKNQFSLKLSSVTAADTAVYYCARQDMTQQTSWGQGTLVTVSSGGGGSGGGGSGGSALQSVLTQPPSASGTPGQ
RVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCA
AWDDSLRNLVFGEGTKLTVLG
No. 67 - >K3+
QVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPNYSPSLKSRATISVDK
SKNQFSLKLSSVTAADTAVYYCARQDMTQQTSWGQGTLVTVSSGEGGSGEGGSGGSALQSVLTQPPSASGTPGQ
RVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCA
AWDDSLRNLVFGEGTKLTVL
No. 68 - >ZA4 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQP
PGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDDK
GGWGQGTLVTVSSGGGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNIGSN
TVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAW
DDSLRSLVFGGGTKLTVLG
No. 69 - >ZA36 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYH
SGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARPSSIWGQGTLVTVSSG
GGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPK
LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRSLVFGGGTK
LTVLG
, CA 02375033 2001-11-27 No. 70 - >ZA14 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHS
GSTNYNPSLKSRVTISVXKSKNQFSLKLSSVTAXDTAVYYCARPSHHAGTHTWGQGTLVT
VSSGGGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPG
TAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLRALVFG
GGTKLTVLG
No. 71 - >Z9 QVQLQESGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGS
TNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARKGLNFGPWGQGTLVTVSSG
GGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNVGSNTVNWYQQLPGTAPK
LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLRSYVFGGGTK
LTVLG
Furthermore, this concerns the amino acid sequences of mimicry peptides against the TF carbohydrate epitope with the following sequence nos. 72 to 96.
(The numbering, e.g. S1, corresponds to a specific isolated clone) No.72:T1 CLREGHFASFC
No.73:T14 CGMLTPAWIKC
No.74:T4 CETFSNLAFLC
No.75:T7 CEGPEIPAFVC
No.76:T3 CESMVEPAWVC
No.77:T15 CTNDIMPPWVC
No.78:T2 CDGLLLPIWAC
No.79:T11 CAGEFVPVWAC
No.80:T16 CDLGLKPAWLC
No.8l:X3 CGPMCSGSCVPQC
No.82:X9 CDAGCNFFCPWRC
No.83:X2 CGPMCSGSCXPQC
Ig No.84:Y8 VWWWQWS
No.85:Y1 MWRPFWL
No.86:Y4 PPWVXHL
No.87:Y9 LIPQWIV
No.88:W4 CTPADMSGC
No.89:W3 CTPADMSGC
No.90:W16 CPSVWMLDLGPC
No.9l:W15 CHGGLTPLC
No.92:W8 CGPMMLWHW
No.93:W5 CTRHIHWGNAHW
No.94:W14 CTPADMSGW
No.95:A1 CFRGGPWWSLC
No.96:A2 CAVRTWVISEC
The invention is explained in more detail by examples of embodiments, however, it is not limited to these.
Examples of embodiments Example 1 Production of hybridome cell line A76-A/C7 and of antibodies After treatment with Neuraminidase (V. cholerae), Balb/c mice were immunized i.p. with a suspension of live human mammacarcinoma cells of the cell line T-47D (Keydar, I. et al., Eur J Cancer, 15:659, 1979) without an adjuvant. The fusion cell line was X63-Ag8.653 (Kearney, J.F. et al., J Immunol 123:1548, 1979). The hybridome method itself was carried out according to standard methods (e. g. Peters, H.H., et al., "Monoklonale Antikorper, Herstellung and Charakterisierung" [Monoclonal Antibodies, Production and Characterization], Berlin 1985;
Friemel, H. "Immunologische Arbeitsmethoden" [Immunological Working Methods], 4th edition, Jena 1991). The specificity analysis of the monoclonal antibodies (mAK) produced by the hybridome cell lines was based on enzyme immunoassays with natural glycoproteins and synthetic peptides and glycopeptides, immunofluorescence analyses with diverse cell lines as well as immune histochemical studies of tissue sections. For the monoclonal antibody A76-A/C7, the epithelial mucin, MUC1, was unequivocally determined as the specific antigen. IgGl,k was determined as the isotope, with a small portion of IgM of the same specificity using a commercial isotyping kit (Pharmingen, San Diego, USA). An epitope mapping within the framework of the ISOBM TD-4 International Workshop on Monoclonal Antibodies against MUC1 (Tumor Biol. 19, Suppl. 1, 1998) defined the epitope as APDTRPAP. Additional studies using synthetic, glycosylated and non-glycosylated peptides showed that the epitope of the monoclonal antibody A76-A/C7 is, to great extent, determined by its conformation:
- The antibody binds only insignificantly to a single unit (a repeat), despite its containing the epitope sequence.
- The binding to non-glycosylated peptides depends on the length of the peptide or, more specifically, on the number of linked repeats (Fig. la). It is known from the literature that the native conformation of the PDTRP
motive only develops with a peptide length of more than 3 repeats (Fontenot, J.D., et al., J Biomol Struct Dyn 13:245, 1995).
- The binding of the monoclonal antibody A76-A/C7 to a single MUCl unit (1 repeat) is greatly increased if it is glycosylated with GalNAc-3GalNAc or Galal-3GalNAc in the area of the epitope at the Thr (Fig. lb~ see also Karsten, U., et al., Cancer res, 58:2541, 1998).
The antibody was cleaned by ammonium sulphate precipitation followed by an affinity chromatography on protein A-Sepharose.
Obtaining human recombinant antibody fragments that mimic the conformation-dependent epitope of the MUC1 from antibody gene libraries using the phage display method Two different synthetic antibody gene libraries were used which represent human single-chain antibody fragments (scFv). One antibody gene library (Griffin 1 Library; http://www.mrc-cpe.cam.ac.uk/-phage/) comprises more than 109 phages with various combinations of the variable regions of the heavy and light chains of human antibodies with, in part, randomized hypervariable regions that are connected by a peptide piece (linker) and are covalently bound to a phage coat protein (pIII). It is derived from a different antibody gene library (Griffiths, A. et al., 1994, EMBO I., 13:3245-3260). The second, smaller gene library comprises scFv with the same framework (single framework library) which were preselected for active folding of the antibody fragments by binding to protein L and protein A (I. Tomlinson., 9th anniversary conference: "Antibody engineering", IBC Conferences, San Diego 1998; I. Tomlinson, 10th anniversary conference: "Antibody engineering" IBC
Conferences, San Diego 1999; Speaker abstract). The first library originates from the Dr. G. Winter Laboratory and the second from the Dr. I. Tomlinson Laboratory (both MRC Centre for Protein Engineering, Cambridge, U.K.). The specific phages were selected in 2-3 rounds (phage panning) using the proteolytic selection method with the helper phage KM13 (Kristensen, P. and Winter, G. Folding & Design, 3:321, 1998). The cleaned monoclonal antibody A76-A/C7 (35 u/ml in 4 ml) was used as the antigen which was immobilized overnight in a test tube (Immunotube, Nunc, Wiesbaden) in PBS at 4 degrees Celsius.
Alternatively, A76-A/C7 was incubated with the phages; the phages bound to the antibodies were obtained through magnet beads with immobilized anti-IgG antibodies (Deutsche Dynal, Hamburg) . After stringent washing steps (up to 20 times PBS/
O.lo Tween20 followed by 20 times PBS), the phages specifically bound during the selection rounds (3 h at RT) were eluted through the tandem repeat (100 ug/ ml; Biosynthan, Berlin-Buch) that was glycosylated with GalNAc in the PDTR, and subsequently treated with Trypsin (proteolytic selection method). Between the selection rounds, the eluted phages were multiplied with helper phages in the bacteria and selected once again.
Obtaining mimicry peptides that mimic the conformation-dependent epitope of the MUCI from peptide gene libraries using the page display method Analogous to the example for generating antiidiotypical antibodies, in multiple selection rounds specifically binding peptides were obtained from a peptide gene library (gene library of Dr. H. Gollasch; Oligino, L. et al., J Biol Chem 272:29046, 1997) that has 107 different short peptides coupled to the phage coat protein pIII. The expressed peptides are randomized nonapeptides that are flanked , and thus circularized, by two cysteins (CX9C), thus increasing the stability and the affinity.
The selection and testing were carried out as described in the generation of the antiidiotypical antibodies. Similarly, additional linear and circular mimicry peptides were obtained with other peptide libraries. These are peptide libraries that were created in the same way as the aforementioned peptide library. The expressed peptides are linear peptides with 7 amino acids and circular peptides with 7 randomized amino acids flanked by two cysteins (CX10C), circular peptides with 10 randomized amino acids flanked by two cysteins (CX10C), and circular peptides with a total of 9 randomized amino acids with two internal and two flanking cysteins (CX3CX3CX3C).
Specificity tests of the mimicry peptides and the antiidiotypical antibody fragments The selected peptides and antibody fragments were tested for their binding to the monoclonal antibody A76-A/C7 in ELISA tests as well as in the form of a negative control with other IgG and IgM monoclonal antibodies. Furthermore, in ELISA tests they were tested for their binding to a series of well characterized MUC1-specific antibodies that differ in their fine specificity.
For the ELISA tests, the form of peptides and antibody fragments coupled to phages was used. The antiidiotypical svFr and the mimicry peptides can be categorized in groups that:
- bind exclusively to A76-A/C7;
- bind to A76-A/C7 and other MUC1-specific antibodies that bind only to either the conformation epitope (in the PDTR region glycosylated MUC1 tandem repeat) (type A) or whose binding is greatly increased through the PDTR
glycosylation of the MUC1 tandem repeat (conformation induction) (type B) - bind to MUCl-specific antibodies that, besides type A and type B, also bind MUCl-specific antibodies which bind glycosylated and unglycosylated MUC1 tandem repeats to the same extent (type D) - bind strongly to MUC1-specific antibodies and that, with regard to the glycosylation of the PDTR region of the MUC1 repeat to A76-A/C7, behave quite the opposite and do not bind the glycosylated MUC1 peptide or bind much less than the non-glycosylated MUC1 peptide (type C). These mimicry peptides or antiidiotypical scFv can also bind to other types of the MUC1-specific antibody.
The mimicry peptides and antiidiotypical antibody fragments were also tested in ELISA inhibition tests to see whether they, in the form of the syntheticized peptides or cleaned scFv (alone or coupled to phages), specifically and concentration-dependently inhibit the binding of A76-A/C7 to the glycosylated MUC1 peptide (in the epitope PDTR with GalNAc glycosylated tandem repeat) and non-glycosylated oligomers of the 20-mer tandem repeats. These tests were carried out with streptavidin-coated micro test plates (BioTeZ, Berlin-Buch) and biotinylated MUC1 peptides (Biosynthan, Berlin-Buch; Fig. lc) as well as with normal ELISA test plates on which the MUC1 peptides were immobilized by drying.
Inbred mice of the Balb/c line were immunized intraperitoneally with mimicry peptides and antiidiotypical antibody fragments in the form of the synthesized peptides or cleaned scFv alone, respectively coupled to the protein KLH or to bacteriophages in PBS, mixed with an incomplete Freund's adjuvant, whereby mixtures of antiidiotypical scFv phages or mimicry peptide phages from the respective groups (see above) were used. Three weeks later a booster of the same preparation, but without the adjuvant, was administered. The booster was repeated after three weeks and ten days later blood samples were taken from the mice. In ELISA tests the serum was tested for antibodies that specifically recognize the conformation-dependent epitope of the MUC1 (test setup as above). The compositions of the antiidiotypical scFv as well as the mimicry peptides trigger a strong reaction against the conformation-dependent epitope of the MUCl.
Design of the DNA vaccines and testing on mice The antiidiotypical scFv were directionally cloned into a DNA
vaccination vector, whereby the scFv were cut out of the phage vector by means of Sfil and NotI and directionally cloned into various DNA vaccination vectors that had previously been split with the same enzymes. A suitable vector for this is the vector pVAC2 (I. Harmer et al., Keystone Symposium "DNA Vaccines", Snowbird, USA, 1999; poster and poster abstract) which, after insertion of the scFv into the DNA vaccination vector, encodes a fusion protein from the antiidiotypical scFv with a tetanus toxoid. The tetanus toxoid has the characteristic of an adjuvant and strengthens the immune response against the fused protein portion (C. King et al., 1998, Nat Medicine 4:1281-86).
The mimicry peptides were also cloned into various DNA vaccine vectors. The cloning occurred according to the well known method of PCR cloning in which, using synthetic primers, the sequences that encode for the mimicry peptides were inserted into the DNA
vaccination vectors. Thus DNA vaccination vectors were also produced on the basis of the pVAC2 which were encoded with the tetanus toxoid for a fusion protein of the mimicry peptide.
The DNA of the vaccination vectors was increased according to well known methods, cleaned and then injected into mice.
Mixtures of DNA vaccination vectors that encode antiidiotypical scFv or mimicry peptides as the fusion protein using the tetanus toxoid and which respectively originate from the various groups with different binding patterns for MUC1-specific antibodies (see above) were used for the immunization. The dose was 50 ug or 200 ug, respectively, of total DNA and administration was intramuscular. Four weeks later a booster of the same preparation was administered and then repeated again after 4 weeks. 10 days later blood samples were taken from the mice. In ELISA tests the serum was tested for antibodies that specifically recognize the conformation-dependent epitope of the MUC1 (test setup as above).
The immunization with the mixtures of DNA vaccine vectors showed a strong humoral immune response against the conformation-dependent epitope of the MUCl and a strong response against the tetanus toxoid in the antiidiotypical scFv as well as the mimicry peptides of the coding DNA vectors.
Vaccines in the tumour challenge model In the mouse tumour challenge model, different mouse tumour cells (3T3 and P815) were used that stably transfect with the cDNA of the transmembrane form of the human MUC1. The MUC1-positive mouse cell lines express the conformation epitope of the MUC1 which was tested in immune binding studies with the A76-A/C7. Several mouse lines were used for the studies (Balb/c, DBA/2 and C57BL/6). After vaccinating the mice according to the prime boost protocol described below, the mice were subcutaneously injected near the peritoneum with 106 to 10' tumour cells in 200 a PBS and the tumour growth (tumour size in mm) was measured over 20 - 30 days.
Prime boost vaccination pattern:
For the immunizations (priming), a combination of DNA
vaccination vectors (encoding for scFv tetanus toxoid or mimicry peptide tetanus toxoid fusion protein) was used with two candidates from each of the 4 different groups of antiidiotypical scFv or mimicry peptides. However, for the booster, the same combinations of antiidiotypical scFv or mimicry peptides were used in incomplete Freund's adjuvants, but in their protein form. To this end, the scFv were cleaned by nickel chelate chromatography according to well known procedures and the mimicry peptides were coupled to KLH according to well known procedures. For the immunization, 50 - 200 ug of total DNA
were administered intramuscularly and for the scFv and the mimicry peptides, 10 - 200 ug were administered intraperitoneally at three week intervals and boosters were administered 2 - 3 times.
As a control, the DNA vaccination vectors were used for a scFv with a specificity against an irrelevant bacterial protein or for an irrelevant peptide (SSGSSSSGS), or their cleaned scFv or the peptide KLH complex. 5 - 10 animals were studied for the different test preparations.
The test showed that a vaccination according to the prime boost protocol prevents the growth of injected MUCl-positive mouse tumour cells or reduces them to a minimal size (0 - 20 mm2 after 20 days). In a subsequent injection with the same tumour cells without transfected MUC1, the same vaccination achieves an average tumour size of over 200 mm2 (after 20 days). The injection of MUC1-positive mouse tumour cell lines into mice without prior vaccination results in strong tumour growth (>200 mm2 after 20 days). An immunization and booster with the proteins of the antiidiotypical scFv or the mimicry peptides coupled to KLH without DNA vaccination vectors results in an immune response against the MUC1 tumour cells, however, the tumour protection is much less than with the prime boost protocol with the DNA vaccination vectors.
The results show that a vaccination with DNA vaccination vectors that encode for antiidiotypical scFv or mimicry peptides provides excellent protection against tumours. This reaction is MUCl-specific. It is far better or even possible compared to vaccination studies with the proteins of the antiidiotypical scFv or mimicry peptides without prior immunization with the corresponding DNA vaccination vectors.
This shows that the vaccines against conformation-dependent antigens according to the invention using DNA vaccination vectors of mimicry structures is a successful form of fighting tumours that carry these conformation-dependent antigens.
Example 2 Production of hybridome cell lines A78-G/A7 and of antibodies In the case of A78-G/A7 (see also Karsten, U. et al., Hybridoma 14:37, 1995), Balb/c mice were immunized intraperitoneally with 100 ug of Asialoglycophorin (Sigma, Diesenhofen) in PBS mixed with Freund's adjuvant. After 24 h, 100 ug of cyclophosphamid in PBS per kg body weight was administered i.p. A booster of 100 ug Asialoglycophorin was administered two weeks later. Each time the fusion cell line was X63-Ag8.653 (Kearney, J.F. et al., J
Immunol 123:1548, 1979). The hybridome method was carried out according to standard methods (e. g. Peter, H.H. et al., "Monoklonale Antikorper, Herstellung and Charakterisierung"
[Monoclonal Antibodies, Production and Characterization], Berlin 1985; Friemel, H. "Immunologische Arbeitsmethoden"
[Immunological Working Methods], 4th edition, Jena 1991). The specificity analysis of the monoclonal antibodies produced by the hybridome cell lines was based on enzyme immunoassays with natural glycoproteins, synthetic peptides and glycopeptides, glycolipids and neoglycolipids and synthetic polyacrylamide-carbohydrate conjugates, absorption analyses of synthetic carbohydrate conjugates (Synsorb, Chembiomed, Edmonton, Canada), immunofluorescence analyses with diverse cell lines as well as immune histochemical studies of tissue sections. For the A78-G/A7, the carbohydrate epitope Thomsen-Friedenreich (TF) that is associated with tumours was unequivocally determined as a specific antigen:
- A78-G/A7 binds exclusively to the disaccharide TF in the a-anomer configuration (TFa; Gall-3GalNAca1-0-Ser/Thr) on natural and synthetic structures as occur naturally only on glycoproteins in the form of a direct 0-glycoside binding to serines or threonines. However TFa, which can occur at the end of glycan chains of glycolipids, as well as other carbohydrate structures, portions of peptides or lipids, are not bound.
- A78-G/A7 binds highly specifically to various cancer cell lines in immunofluorescence studies and to various cancers in histochemical studies. (Cao, Y. et al., Histochem Cell Biol 106:197, 1996; Cao, Y. et al., Cancer 76:1700, 1995; Cao, Y.
et al., Virchows Arch 431:159, 1997; Karsten, U. et al., Hybridoma 14:37, 1995.
- For A78-G/A7, IgM, k, was determined as the isotype using a commercial isotyping kit (Pharmingen, San Diego, USA).
A78-G/A7 was cleaned using an ammonium sulphate precipitation followed by an affinity chromatography on a proteinG affinity matrix for cleaning undesirable IgG antibodies from the calf serum and, finally, with an affinity chromatography using a goat-anti-mouse-Ig affinity matrix (Perzellulose, BioTeZ, Berlin-Buch) (Dr. G. Butschak).
Production of human recombinant antibody fragments against the Thomsen-Friedenreich antigen from antibody libraries using the phage display method Two different synthetic antibody gene libraries were used which represent human single-chain antibody fragments (scFv). One antibody gene library comprises more than 101° phages with various combinations of the variable regions of the heavy and light chains of human antibodies with, in part, randomized hypervariable regions that are connected by a peptide piece (linker) and are covalently bound to a phage coat protein (pIII). It is derived from a different antibody gene library (Griffiths, A. et al., 1994, EMBO I., 13:3245-3260). The second, smaller gene library comprises scFv which were preselected for active folding of the antibody fragments. The first library originates from the Dr. G. Winter Laboratory and the second from the Dr. I. Tomlinson Laboratory (both MRC Centre for Protein Engineering, Cambridge, U.K.). The specific phages were selected in 2-3 rounds (phage panning) using the proteolytic selection method with the helper phage KM13 (Kristensen, P. and Winter, G. Folding and Design, 3:321, 1998). The cleaned monoclonal antibody A78-G/A7 (35 u/ml in 4 ml) was used as the antigen which was immobilized overnight in a test tube (Immunotube, Nunc, Wiesbaden) in PBS at 4 degrees Celsius.
Alternatively, the cleaned antibody was incubated with the phages; the phages bound to the antibodies were obtained through magnet beads with immobilized anti-IgM antibodies (Deutsche Dynal, Hamburg). After stringent washing steps (up to 20 times PBS/ O.lo Tween20 followed by 20 times PBS), the phages specifically bound during the selection rounds (3 h at RT) were specifically eluted through the TFa-carrying glycoprotein asialglycophorin (100 - 165 ug/ ml) and, in part, subsequently treated with Trypsin (proteolytic selection method). Between the selection rounds, the eluted phages were multiplied with helper phages in the bacteria and selected once again. 2 to 3 selection rounds were carried out.
Identification of peptides using a peptide gene library that specifical3y mimics the Thomsen-Friedenreich antigen Analogous to the example for generating antiidiotypical antibodies, in multiple selection rounds specifically binding peptides were obtained from a peptide gene library (Oligino, L.
et al., J Biol Chem 272:29046, 1997) that has 10' different short peptides coupled to the phage coat protein pIII (in cooperation with Dr. H. Gollasch Robert-Rossle-Klinik, Berlin-Buch). The expressed peptides are randomized nonapeptides that are flanked, and thus circularized, thereby increasing the stability and the affinity. The selection and testing were carried out as described in the generation of the antiidiotypical antibodies.
Specificity testing of the mimicry peptides and antiidiotypical antibody fragments The selected peptides and antibody fragments were tested in ELISA tests for their binding to the TF-specific antibodies and to the plant lectin PNA (peanut agglutinin, Arachis hypogaea lectin; Sigma) which also , if not exclusively, binds the Thomsen-Friedenreich antigen, as well as for comparison with other IgM and IgG antibodies. To this end, the form of peptides and antibody fragments that are coupled to phages were used which were cleaned beforehand through a polyethylene glycol precipitation in 96-well plates. The potential mimicry peptides and antiidiotypical antibody fragments were then studied in ELISA inhibition tests as to whether they specifically inhibit the binding of A78-G/A7 and/or other TF-recognizing antibodies and lectins to the disaccharide TFa. To this end, the TFa-carrying glycoprotein asialglycophorin was immobilized on ELISA
plates by drying, and the binding of the monoclonal antibodies and lectins was concentration-dependently inhibited by the mimicry peptides or antiidiotypical antibody fragments in the form of the synthetic peptides or cleaned scFv alone or coupled to phages (Fig. 2).
~
Inbred mice of the Balb/c and NMRI lines were immunized intraperitoneally with mimicry peptides and antiidiotypical antibody fragments in the form of the synthesized peptides or cleaned scFv alone, respectively coupled to the protein KLH or to bacteriophages in PBS, mixed with a complete Freund's adjuvant. Three weeks later a booster of the same preparation, but without the adjuvant, was administered. The booster was repeated after three weeks and ten days later blood samples were taken taken from the mice. In ELISA tests the serum was tested for antibody bindings against the Thomsen-Friedenreich antigen.
Vaccination with TF-mimicking peptides in the mouse tumour model Cell culture: The mouse colon cancer cell line C-26 was kept in the medium RPMI 1640 with the addition of loo foetal calf serum.
Tumour model: 105 cells of the syngene colon cancer cell line C-26 were transplanted subcutaneously into mice of the Balb/c line in two variants: a) untreated and b) pretreated (TF-positive) with neuraminidase from V. cholerae (Serva, Heidelberg). At weekly intervals the tumour size was determined externally.
After 3 weeks the animals were killed and the livers removed in order to determine the number of visible metastases on the surface of the liver.
Vaccination: The vaccination of the mice was begun 6 weeks prior to the tumour transplant. The phage preparation or the cleaned scFv (as well as corresponding controls) were emulgated 1:1 with an incomplete Freund adj uvants and inj ected i . p . Four weeks later a booster was administered (without adjuvant). After two more weeks, the tumour transplant (tumour challenge) was carried out with untreated and C-26 cells treated with neuraminidase.
Results: The present results with three of the aforementioned antiidiotypical scFv showed that the initial rate of tumours in the C-26 cells treated with neuraminidase can be significantly reduced through vaccination (to 3 - 16% of the control; control:
1000 initial rate). Furthermore, the number of liver metastases in the vaccinated animals approximately corresponded to that of the animals that were transplanted with untreated (TF-negative) C-26 cells (ca. 2 per liver), whereas the unvaccinated control animals with TF-positive C-26 cells had 5 - 9 metastases per liver.
Legends for the Figures:
Fig. lc:
Inhibition of the A76-A/C7 binding to the MUC1 glycopeptide (Biotin-Ahx-APPAHGVTSAPD-Thr(a-D-GalNAc)-RPAPGSTAPPAHGVTSA) through scFv phages. The MUC1 glycopeptide was immobilised on the streptavidin ELISA plate (5 ng/well) and subsequently blocked with 30% FKS in RPMI. The culture supernatant of the A76-A/C7 (diluted 1:80 ) was preincubated for one hour with the scFv phages that were cleaned through a polyethylene glycol precipitation in the indicated concentrations (percent by volume of adjusted phage solutions in PBS) and subsequently placed on the MUC1 glycopeptide plate for 2 hours. Verification occurred via an anti-mouse-POD antibody (Dako). The scFv phages Q6, Q7 and Q8 are examples of antiidiotypical scFv, whereas Q4 and Q10 are examples of control scFv that bind the A78-A/C7 but are not, however, antiidiotypical scFv.
Figure 2:
Inhibition of the A78-G/A7 binding to Asialoglycophorin through scFv phages. The Asialoglycophorin (A-GP) was immobilized on the ELISA plate by drying (25 ng/well) and subsequently blocked with 30% FKS in RPMI. The culture supernatant of the A78-G/A7 (diluted 1:20 ) was preincubated for one hour with the scFv phages that were cleaned through a polyethylene glycol precipitation in the indicated concentrations (percent by volume of adjusted phage solutions in PBS) and subsequently placed on the A-GP plate for 2 hours. Verification occurred via an anti-mouse-POD antibody (Dako). The scFv phages P9, P13, P16, P3 and K3 are examples of antiidiotypical scFv, whereas P8 and Ql are examples of control scFv, of which P8 binds the A78-G/A7 but is not, however, an antiidiotypical scFv, and Ql is a phage that does not bind A78-G/A7.
Claims (26)
1. Vaccines against conformation-dependent antigens, characterized by a. a DNA that encodes that region of an antiidiotypical antibody (Ab2), of an antiidiotypical antibody fragment or of another peptide, which specifically binds the binding location of an antibody (Ab1) or of a molecule that specifically binds the antigen and immunologically mimics the original antigen, the epitope being wholly or partially conformation-dependent and having an immunogenic structure that is not defined by a simple sequence of amino acids of the antigen's primary sequence, but rather by a specific spatial conformation of amino acids, and the DNA is applied in the form of naked DNA, linear or circular, and/or using a viral vector with or without an adjuvant, or b. an antibody, an antibody fragment or a peptide that immunologically mimics the conformation-dependent antigen, or c. a combination of substances from a and b.
2. Vaccines according to Claim 1, characterized in that the immunogenic structures are defined by a specific spatial conformation of amino acids that developed, for instance, through the interaction of amino acids that are not adjacent in the primary sequence of the antigen, or are caused by the development of a secondary or higher structural arrangement due to an interaction of amino acids from proteins of a protein complex, or by the modification of the primary structures, for instance through glycosylation or phosphorylation.
3. Vaccines against antigens that are not, or not exclusively, proteins or peptides, characterized by a. a DNA that encodes that region of an antiidiotypical antibody (Ab2), of an antiidiotypical antibody fragment or of another peptide, which specifically binds the binding location of an antibody (Ab1) or of a molecule that binds the antigen and immunologically mimics the original antigen, the antigen being substances whose relevant epitopes are not proteins or peptiepitopes, however, who do have an immunological structure and the DNA is applied in the form of naked DNA, linear or circular, and/or using a viral vector with or without an adjuvant, or b. an antibody, an antibody fragment or a peptide that immunologically mimics the antigen that is not, or not exclusively, a protein or a peptide, or c. a combination of the substances in a and b.
4. Vaccines according to Claim 3, characterized in that immunological structures of the relevant epitope are preferably glycostructures, combined carbohydrate protein epitopes, lipids, glycolipids or synthetic structures.
5. Vaccines according to Claims 1 to 4, characterized in that there are peptides, linear or circular, for instance by inserting cysteins at suitable locations.
6. Use of a vaccine according to Claims 1 to 5 for immunization by means of DNA and/or the antibodies, antibody fragments (antiidiotypical antibodies) or peptides (mimicry peptides) that immunologically mimic the antigen.
7. Use of the vaccines according to Claim 6, characterized by formulations of these protein structures which are suitable for vaccines, either by administering the DNA that encodes them according to 1a or 3a, or by administering the structures alone, such as peptides, inverse peptides or retroinverse peptides in the form of a chemical coupling to proteins, such as Keyhole limpet hemocyanin (KLH) in the form of bacteriophages as fusion proteins with phage coat proteins on their surface, in the form of a fusion protein on the surface of other viruses or attenuated biological carriers, or by loading dendritic cells according to well known processes, either in combination with suitable adjuvants or immune-stimulating molecules such as cytokines, which can also be administered in the form of a DNA that encodes them.
8. Use of the vaccines according to Claims 6 and 7, characterized by a combination of the DNA and the protein structures in a suitable formulation.
9. Use of the vaccines according to Claims 1, 2, 5, 6, 7 and 8 against tumour-associated conformation-dependent antigens.
10. Use of the vaccines according to Claims 3 to 8 against tumour-associated antigens that are not, or not exclusively, proteins or peptides.
11. Use of the vaccines according to Claims 1, 2, 5, 6, 7 and 8 against conformation-dependent antigens of infectious disease pathogens such as prions, viruses, bacteria, parasites.
12. Use of the vaccines according to Claims 3 to 8 against antigens of infectious disease pathogens such as prions, viruses, bacteria, parasites that are not, or not exclusively, proteins or peptides.
13. Use of the vaccines according to Claims 1 to 8 against other infectious or non-infectious diseases in the fields of human and veterinary medicine.
14. Process for the production of a vaccine against conformation-dependent antigens according to one or more of Claims 1, 2 or 5 on the basis of immunologically mimicking structures in the form of antiidiotypical antibodies, antiidiotypical antibody fragments or mimicry peptides or DNA sequences resulting therefrom, characterized in that:
a. using the hybridome method, monoclonal antibodies (Ab 1) against conformation-dependent antigens according to Claim 1 and antiidiotypical antibodies (Ab2, type b), which immunologically mimic the antigen according to Claims 1 and 2, b. using the phage display method or the ribosome display method, from genome, hybrid, semisynthetic or synthetic antibody libraries as well as from gene libraries of immunized or non-immunized donors, recombinant antibody fragments (Ab1) against conformation-dependent antigens or, using idiotypical antibodies or antibody fragments that specifically recognize the conformation-dependent antigen, recombinant antiidiotypical antibody fragments (Ab2) that immunologically mimic the antigen according to Claims 1, 2 and 5, c. using the phage display method or the ribosome display method and using substances (e. g. receptors) that specifically recognize the conformation-dependent antigen, from genome, hybrid, semisynthetic or synthetic antibody libraries as well as from gene libraries of immunized or non-immunized donors, recombinant antibody fragments that immunologically mimic the conformation-dependent antigen according to Claims 1, 2 and 5, d. using the phage display method or the ribosome display method and using idiotypical antibodies or antibody fragments that specifically recognize the conformation-dependent antigen, from synthetic peptide libraries, linear or circular peptides that bind the antigen-binding regions of the conformation-specific antibodies (Ab1) according to Claim 1 and, thus, immunologically mimic the antigen according to Claims 1, 2 and 5, e. using the phage display method or the ribosome display method and using substances (e. g. receptors) that specifically recognize the target antigen, from synthetic peptide gene libraries, linear or circular peptides that bind the antigen-binding regions of the conformation-specific antibodies (Ab1) according to Claim 1 and, thus, immunologically mimic the antigen according to Claims 1, 2 and 5, are produced or selected, and a DNA according to Claim 1 that corresponds to the antibodies or peptides according to a-e or suitable partial peptides or derived peptides (for instance through circularization, mutations, in the form of inverse or retroinverse peptides) or repetitive designs is produced according to well known processes.
a. using the hybridome method, monoclonal antibodies (Ab 1) against conformation-dependent antigens according to Claim 1 and antiidiotypical antibodies (Ab2, type b), which immunologically mimic the antigen according to Claims 1 and 2, b. using the phage display method or the ribosome display method, from genome, hybrid, semisynthetic or synthetic antibody libraries as well as from gene libraries of immunized or non-immunized donors, recombinant antibody fragments (Ab1) against conformation-dependent antigens or, using idiotypical antibodies or antibody fragments that specifically recognize the conformation-dependent antigen, recombinant antiidiotypical antibody fragments (Ab2) that immunologically mimic the antigen according to Claims 1, 2 and 5, c. using the phage display method or the ribosome display method and using substances (e. g. receptors) that specifically recognize the conformation-dependent antigen, from genome, hybrid, semisynthetic or synthetic antibody libraries as well as from gene libraries of immunized or non-immunized donors, recombinant antibody fragments that immunologically mimic the conformation-dependent antigen according to Claims 1, 2 and 5, d. using the phage display method or the ribosome display method and using idiotypical antibodies or antibody fragments that specifically recognize the conformation-dependent antigen, from synthetic peptide libraries, linear or circular peptides that bind the antigen-binding regions of the conformation-specific antibodies (Ab1) according to Claim 1 and, thus, immunologically mimic the antigen according to Claims 1, 2 and 5, e. using the phage display method or the ribosome display method and using substances (e. g. receptors) that specifically recognize the target antigen, from synthetic peptide gene libraries, linear or circular peptides that bind the antigen-binding regions of the conformation-specific antibodies (Ab1) according to Claim 1 and, thus, immunologically mimic the antigen according to Claims 1, 2 and 5, are produced or selected, and a DNA according to Claim 1 that corresponds to the antibodies or peptides according to a-e or suitable partial peptides or derived peptides (for instance through circularization, mutations, in the form of inverse or retroinverse peptides) or repetitive designs is produced according to well known processes.
15. Process for the production of vaccines against antigens according to Claims 3, 4 and 5 on the basis of immunologically mimicking structures in the form of antiidiotypical antibody fragments or mimicry peptides or DNA sequences resulting therefrom, characterized in that:
a. using the phage display method or the ribosome display method, from genome, from genome, hybrid, semisynthetic or synthetic antibody libraries as well as from gene libraries of immunized or non-immunized donors, recombinant antibody fragments (Ab1) against antigens that primarily are not proteins or peptides, or using idiotypical antibodies or antibody fragments that specifically recognize the conformation-dependent antigen, recombinant antiidiotypical antibody fragments (Ab2) that immunologically mimic the antigen according to Claims 3, 4 and 5, b. using the phage display method or the ribosome display method and using substances such as lectins, receptors, peptides that specifically recognize the target antigen, from genome, hybrid, semisynthetic or synthetic antibody libraries as well as from gene libraries of immunized or non-immunized donors, recombinant antibody fragments that immunologically mimic the target antigen according to Claims 3, 4 and 5, c. using the phage display method or the ribosome display method, from synthetic peptide gene libraries, linear or circular peptides that bind according to Claims 3 and 4 the antigen-binding regions of the antibodies (Ab1) against antibodies that are not, or not exclusively, proteins or peptides and, thus, immunologically mimic the antigen according to Claims 3, 4 and 5, d. using the phage display method or the ribosome display method and using substances such as lectins, receptors, peptides that specifically recognize the target antigen, from synthetic peptide gene libraries, linear or circular peptides that immunologically mimic the target antigen according to Claims 3, 4 and 5, are produced or selected, and a DNA according to Claim 3 that corresponds to the antibodies or peptides according to a-d or suitable partial peptides or derived peptides (for instance through circularization, mutations, in the form of inverse or retroinverse peptides) or repetitive designs is produced according to well known processes.
a. using the phage display method or the ribosome display method, from genome, from genome, hybrid, semisynthetic or synthetic antibody libraries as well as from gene libraries of immunized or non-immunized donors, recombinant antibody fragments (Ab1) against antigens that primarily are not proteins or peptides, or using idiotypical antibodies or antibody fragments that specifically recognize the conformation-dependent antigen, recombinant antiidiotypical antibody fragments (Ab2) that immunologically mimic the antigen according to Claims 3, 4 and 5, b. using the phage display method or the ribosome display method and using substances such as lectins, receptors, peptides that specifically recognize the target antigen, from genome, hybrid, semisynthetic or synthetic antibody libraries as well as from gene libraries of immunized or non-immunized donors, recombinant antibody fragments that immunologically mimic the target antigen according to Claims 3, 4 and 5, c. using the phage display method or the ribosome display method, from synthetic peptide gene libraries, linear or circular peptides that bind according to Claims 3 and 4 the antigen-binding regions of the antibodies (Ab1) against antibodies that are not, or not exclusively, proteins or peptides and, thus, immunologically mimic the antigen according to Claims 3, 4 and 5, d. using the phage display method or the ribosome display method and using substances such as lectins, receptors, peptides that specifically recognize the target antigen, from synthetic peptide gene libraries, linear or circular peptides that immunologically mimic the target antigen according to Claims 3, 4 and 5, are produced or selected, and a DNA according to Claim 3 that corresponds to the antibodies or peptides according to a-d or suitable partial peptides or derived peptides (for instance through circularization, mutations, in the form of inverse or retroinverse peptides) or repetitive designs is produced according to well known processes.
16. Process according to Claims 14 and 15, characterized in that vaccines are produced according to Claims 1 to 5.
17. Process according to Claims 14 and 16, characterized in that a vaccine against the immune-dominant epitope (PDTR) of the MUC1 is produced, whose conformation (which is important for immunogenicity) is developed through the glycosylation of the Thr in the epitope PDTR.
18. Process according to Claims 15 and 16, characterized in that a vaccine is produced against the tumour-associated glycostructures Core-1 structure (GalNAc.beta.1-3-GalNAc.alpha.1), Tn or Sialyl-Tn.
19. Human antiidiotypical antibody fragments against the MUC1 conformation epitope with the sequence nos. 1 to 31 as well as protein sequences and partial sequences derived therefrom and having the same characteristics.
20. DNA sequences that encode according to Claim 19 the fragments and proteins or partial sequences derived therefrom and having the same characteristics.
21. Amino acid sequences of mimicry peptides against the MUC1 conformation epitope with the sequence nos. 32 to 47 as well as peptide sequences and partial sequences derived therefrom and having the same characteristics.
22. DNA sequences that encode according to Claim 21 the amino acid sequences and peptides or partial sequences derived therefrom and having the same characteristics.
23. Antiidiotypical antibody fragments against the TF antigen with the sequence nos. 48 to 71 as well as protein sequences and partial sequences derived therefrom and having the same characteristics.
24. DNA sequences that encode according to Claim 23 the fragments and proteins or partial sequences derived therefrom and having the same characteristics.
25. Amino acid sequences of mimicry peptides against the TF
carbohydrate epitope with the sequence nos. 71 to 96 as well as peptide sequences and partial sequences derived therefrom and having the same characteristics.
carbohydrate epitope with the sequence nos. 71 to 96 as well as peptide sequences and partial sequences derived therefrom and having the same characteristics.
26.DNA sequences that encode according to Claim 25 the amino acid sequences and peptides or partial sequences derived therefrom and having the same characteristics.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
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| DE19924405 | 1999-05-27 | ||
| DE19924405.7 | 1999-05-27 | ||
| DE19943016.0 | 1999-09-09 | ||
| DE19943016 | 1999-09-09 | ||
| PCT/DE2000/001809 WO2000073430A2 (en) | 1999-05-27 | 2000-05-29 | Vaccines against conformation-dependent antigens and against antigens that are not or are not only proteins or peptides |
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| Publication Number | Publication Date |
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| CA2375033A1 true CA2375033A1 (en) | 2000-12-07 |
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| CA002375033A Abandoned CA2375033A1 (en) | 1999-05-27 | 2000-05-29 | Vaccines against conformation-dependent antigens and against antigens that are not or are not only proteins or peptides |
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| Country | Link |
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| EP (1) | EP1181058A2 (en) |
| AU (1) | AU6424300A (en) |
| CA (1) | CA2375033A1 (en) |
| DE (1) | DE10027695A1 (en) |
| WO (1) | WO2000073430A2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7658924B2 (en) | 2001-10-11 | 2010-02-09 | Amgen Inc. | Angiopoietin-2 specific binding agents |
| US8030025B2 (en) | 2008-02-20 | 2011-10-04 | Amgen Inc. | Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof |
| US8642276B2 (en) * | 2002-07-22 | 2014-02-04 | Glycotope Gmbh | Method for the production of an immunostimulating mucin (MUC1) |
| US20140206021A1 (en) * | 2002-12-03 | 2014-07-24 | North Carolina State University | Prion protein ligands and methods of use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6608182B1 (en) | 1994-03-08 | 2003-08-19 | Human Genome Sciences, Inc. | Human vascular endothelial growth factor 2 |
| US7153827B1 (en) | 1994-03-08 | 2006-12-26 | Human Genome Sciences, Inc. | Vascular endothelial growth factor 2 and methods of use |
| US6040157A (en) | 1994-03-08 | 2000-03-21 | Human Genome Sciences, Inc. | Vascular endothelial growth factor 2 |
| US7109308B1 (en) | 1994-03-08 | 2006-09-19 | Human Genome Sciences, Inc. | Antibodies to human vascular endothelial growth factor 2 |
| US5932540A (en) | 1994-03-08 | 1999-08-03 | Human Genome Sciences, Inc. | Vascular endothelial growth factor 2 |
| DK0751992T3 (en) | 1994-03-08 | 2006-03-06 | Human Genome Sciences Inc | Karendothelial growth factor 2 |
| US7186688B1 (en) | 1994-03-08 | 2007-03-06 | Human Genome Sciences, Inc. | Methods of stimulating angiogenesis in a patient by administering vascular endothelial growth factor 2 |
| US7223724B1 (en) | 1999-02-08 | 2007-05-29 | Human Genome Sciences, Inc. | Use of vascular endothelial growth factor to treat photoreceptor cells |
| WO2002011769A1 (en) | 2000-08-04 | 2002-02-14 | Human Genome Sciences, Inc. | Vascular endothelial growth factor 2 |
| US7402312B2 (en) | 2001-04-13 | 2008-07-22 | Human Genome Sciences, Inc. | Antibodies to vascular endothelial growth factor 2 (VEGF-2) |
| DE60236646D1 (en) * | 2001-04-13 | 2010-07-22 | Human Genome Sciences Inc | Anti-VEGF-2 antibodies |
| US7521053B2 (en) | 2001-10-11 | 2009-04-21 | Amgen Inc. | Angiopoietin-2 specific binding agents |
| JP2005289809A (en) | 2001-10-24 | 2005-10-20 | Vlaams Interuniversitair Inst Voor Biotechnologie Vzw (Vib Vzw) | Mutant heavy-chain antibody |
| EP1539235A2 (en) | 2002-07-01 | 2005-06-15 | Human Genome Sciences, Inc. | Antibodies that specifically bind to reg iv |
| GB0324265D0 (en) * | 2003-10-16 | 2003-11-19 | Medical Res Council | Peptide |
| JP5020808B2 (en) * | 2004-03-29 | 2012-09-05 | ザ・ユニバーシティ・コート・オブ・ザ・ユニバーシティ・オブ・アバディーン | Specific binding elements for synaptophysin |
| EP1871882A1 (en) * | 2005-03-25 | 2008-01-02 | GlycArt Biotechnology AG | Antigen binding molecules directed to mcsp and having increased fc receptor binding affinity and effector function |
| EP2949668B1 (en) | 2005-05-18 | 2019-08-14 | Ablynx N.V. | Improved nanobodies tm against tumor necrosis factor-alpha |
| DE102005023617A1 (en) | 2005-05-21 | 2006-11-23 | Aspre Ag | Method for mixing colors in a display |
| CN1301267C (en) * | 2005-06-21 | 2007-02-21 | 中国人民解放军军事医学科学院附属医院 | A simulated epitope peptide of MUC1 mucoprotein and its encoding DNA and use thereof |
| NZ587506A (en) * | 2008-01-25 | 2012-09-28 | Univ Aarhus | Selective exosite inhibition of papp-a activity against igfbp-4 |
| NZ623273A (en) | 2008-12-05 | 2015-09-25 | Abraxis Bioscience Llc | Sparc binding scfvs |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH04304897A (en) * | 1991-04-01 | 1992-10-28 | Kyowa Hakko Kogyo Co Ltd | Anti-idiotype monoclonal antibody |
| DE19627352A1 (en) * | 1996-06-27 | 1998-01-02 | Max Delbrueck Centrum | Vaccine against carbohydrate antigens |
| WO1999040433A1 (en) * | 1998-02-04 | 1999-08-12 | The Trustees Of The University Of Pennsylvania | Peptide mimotopes of carbohydrate antigens |
| GB9808327D0 (en) * | 1998-04-20 | 1998-06-17 | Chiron Spa | Antidiotypic compounds |
-
2000
- 2000-05-29 AU AU64243/00A patent/AU6424300A/en not_active Abandoned
- 2000-05-29 CA CA002375033A patent/CA2375033A1/en not_active Abandoned
- 2000-05-29 WO PCT/DE2000/001809 patent/WO2000073430A2/en active Search and Examination
- 2000-05-29 DE DE10027695A patent/DE10027695A1/en not_active Withdrawn
- 2000-05-29 EP EP00951201A patent/EP1181058A2/en not_active Withdrawn
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| US9487567B2 (en) | 2002-07-22 | 2016-11-08 | Glycotope Gmbh | Method for the production of an immunostimulating mucin (MUC1) |
| US20140206021A1 (en) * | 2002-12-03 | 2014-07-24 | North Carolina State University | Prion protein ligands and methods of use |
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| US8030025B2 (en) | 2008-02-20 | 2011-10-04 | Amgen Inc. | Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof |
| US8221749B2 (en) | 2008-02-20 | 2012-07-17 | Amgen Inc. | Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof |
| US10336820B2 (en) | 2008-02-20 | 2019-07-02 | Amgen Inc. | Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof |
Also Published As
| Publication number | Publication date |
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| WO2000073430A2 (en) | 2000-12-07 |
| WO2000073430A3 (en) | 2001-03-29 |
| DE10027695A1 (en) | 2001-04-19 |
| AU6424300A (en) | 2000-12-18 |
| EP1181058A2 (en) | 2002-02-27 |
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